Casein protein hydrolysate having anti-inflammatory efficacy and methods of making and use

Abstract

The application provides a lacto-casein hydrolysate with anti-inflammatory effect and a preparation method and application thereof.The lacto-casein hydrolysate of the application comprises a peptide segment FAWPQYLK, a peptide segment LPW, a peptide segment MPLW and a peptide segment PLW, can reduce the release of pro-inflammatory cytokines, such as IL-1beta, IL-2, IL-6 and TNF-alpha, and improve the release of anti-inflammatory cytokines, such as IL-10, so as to reduce the cell inflammatory response, and further achieve the anti-inflammatory effect.

Description

TECHNICAL FIELD

[0001] The present application relates to biotechnology, and in particular to a milk casein hydrolysate with anti-inflammatory effect and a preparation method and application thereof. BACKGROUND

[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 secretes various cytokines to regulate inflammation to stimulate, recruit and expand immune cells, leading to the occurrence of inflammatory response. Inflammation is a defense mechanism against harmful substances invading life, allowing damaged tissues to repair and heal. However, if the inflammatory response is too strong or lasts too long, it may also cause a series of damage to the body, and even endanger life in severe cases. For example, inflammation can damage the vascular endothelium, and the human immune mechanism can repair the damaged tissue, which causes the vascular endothelium to thicken, leading to easier deposition of lipids on the vascular wall, thereby inducing atherosclerosis.

[0003] Bioactive peptides are composed of natural amino acids, which are usually degraded into harmless metabolites in the body, so they have low toxicity and high safety. Bioactive peptides are similar to natural proteins and enzymes in the human body, usually have good biocompatibility, and reduce the risk of immunogenicity and allergic reactions. Bioactive peptides have small molecular weight and clear characteristic structure, which are beneficial to direct absorption and utilization by the body, and have the advantages of strong solubility and strong stability. Therefore, in order to effectively inhibit inflammatory response, it is urgent to find a bioactive peptide with anti-inflammatory effect. SUMMARY

[0004] The present application provides a milk casein hydrolysate which can reduce the release of IL-1β, IL-2 and IL-6 to reduce cell inflammatory response.

[0005] The present application provides a preparation method of a milk casein hydrolysate, which is simple and fast to operate and convenient for large-scale production.

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

[0007] The present application 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 milk casein hydrolysate as described above, wherein the content of the peptide segment FAWPQYLK is ≥1.50% based on the mass of the milk casein hydrolysate; and / or,

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

[0010] a content of the peptide segment MPLW is ≥ 0.05% based on the mass of the milk casein hydrolysate; and / or,

[0011] a content of the peptide segment PLW is ≥ 0.05% based on the mass of the milk casein hydrolysate.

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

[0013] a mass content of the peptides with a molecular weight ≤ 10000 Da in the milk casein hydrolysate is ≥ 80%; and / or,

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

[0015] a mass content of the peptides with a molecular weight < 1000 Da in the milk casein hydrolysate is ≥ 30%.

[0016] The milk casein hydrolysate as described above, wherein the milk casein hydrolysate is obtained after subjecting a milk casein raw material to enzymatic treatment;

[0017] The enzymatic treatment comprises using trypsin and streptomyces protease for enzymolysis.

[0018] The present application provides a preparation method of a milk casein hydrolysate, comprising the following steps:

[0019] subjecting a milk casein raw material to enzymatic treatment to obtain a milk casein hydrolysate;

[0020] The enzymatic treatment comprises using trypsin and streptomyces protease for enzymolysis.

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

[0022] the addition amount of the trypsin is 0.10-0.35wt% based on per gram of the milk casein raw material; and / or,

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

[0024] the addition amount of the streptomyces protease is 0.05-0.32wt% based on per gram of the milk casein raw material.

[0025] The preparation method as described above, wherein the enzymolysis treatment conditions include: an enzymolysis temperature of 40-55℃, an enzymolysis time of 2.0-3.5 h, or an enzymolysis pH value of 7.5-8.2.

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

[0027] The present application provides the lacto-casein hydrolysate as described above, and / or the lacto-casein hydrolysate prepared by the preparation method as described above for use in preparing related products with anti-inflammatory efficacy.

[0028] The present application provides a lacto-casein hydrolysate, which includes peptide segment FAWPQYLK, peptide segment LPW, peptide segment MPLW and peptide segment PLW, can reduce the release of pro-inflammatory cytokines, such as IL-1β, IL-2, IL-6 and TNF-α, and increase the release of anti-inflammatory cytokines, such as IL-10, so as to reduce the cell inflammatory response and achieve anti-inflammatory efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The figure is the cell factor content detection result graph of the peptide segment FAWPQYLK group in Example 4.

[0030] Figure 2 The figure is the cell factor content detection result graph of the peptide segment LPW group in Example 4.

[0031] Figure 3 The figure is the cell factor content detection result graph of the peptide segment MPLW group in Example 4.

[0032] Figure 4 The figure is the cell factor content detection result graph of the peptide segment PLW group in Example 4.

[0033] Figure 5 The figure is the cell factor TNF-α content detection result graph of the lacto-casein hydrolysate group in Example 4.

[0034] Figure 6 The figure is the cell factor IL-10 content detection result graph of the lacto-casein hydrolysate group in Example 4.

[0035] Figure 7 The figure is the cell factor IL-6 content detection result graph of the lacto-casein hydrolysate group in Example 4.

[0036] Figure 8 The figure is the cell factor IL-2 content detection result graph of the lacto-casein hydrolysate group in Example 4.

[0037] Figure 9Figure for the results of the cytokine IL-1β content detection of the milk casein hydrolysate group in Example 4. DETAILED DESCRIPTION

[0038] In order for those skilled in the art to better understand the solutions of the present application, the present application is further described in detail below. The following specific embodiments are only used to describe the principles and characteristics of the present application, and the examples are only used to explain the present application, and do not limit the scope of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[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 secretes various cytokines to regulate inflammation to stimulate, recruit and expand immune cells, leading to the occurrence of inflammatory response. Inflammation is a defense mechanism against harmful substances invading life, allowing damaged tissues to repair and heal. However, 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 even endanger life in severe cases.

[0040] Therefore, in order to regulate pro-inflammatory cytokines and anti-inflammatory cytokines, and effectively inhibit inflammatory response, the present application provides a milk casein hydrolysate, which comprises a peptide segment FAWPQYLK, a peptide segment LPW, a peptide segment MPLW and a peptide segment PLW.

[0041] The peptide segment FAWPQYLK is an octapeptide peptide segment (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-terminal to C-terminal. The peptide segment LPW is a tripeptide peptide segment (Leu-Pro-Trp) composed of leucine (Leu, L), proline (Pro, P), and tryptophan (Trp, W) from N-terminal to C-terminal. The peptide segment MPLW is a tetrapeptide peptide segment (Met-Pro-Leu-Trp) composed of methionine (Met, M), proline (Pro, P), leucine (Leu, L), and tryptophan (Trp, W) from N-terminal to C-terminal. The peptide segment PLW is a tripeptide peptide segment (Pro-Leu-Trp) composed of proline (Pro, P), leucine (Leu, L), and tryptophan (Trp, W) from N-terminal to C-terminal.

[0042] It has been verified by experiments that the peptide segment FAWPQYLK can significantly reduce the release of cytokines IL-1β, IL-2, IL-6 and TNF-α; the peptide segment 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 segment 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; and the peptide segment 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 alpha (TNF-α) belong to cytokines (Cytokine, CK). Cytokines are a class of small molecular proteins with wide biological activity, which are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and some 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 the function they play in the regulation of inflammatory response. Anti-inflammatory cytokines include IL-10, which has a biological effect of inhibiting inflammatory response and helps to resolve inflammation and recover from acute stage 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 response, promote the growth, activation, differentiation and homing of immune cells to the infection site, and help the occurrence and spread of autoimmune inflammation. Therefore, the peptide segment FAWPQYLK, the peptide segment LPW, the peptide segment MPLW and the peptide segment PLW can all reduce the release of pro-inflammatory cytokines and increase the release of anti-inflammatory cytokines to reduce the inflammatory response of cells and achieve anti-inflammatory effect.

[0043] The casein protein hydrolysate is a hydrolysate of casein protein, which is obtained by directional enzyme cutting and specific small peptide separation technology on casein protein. The casein protein hydrolysate not only has the original excellent properties of casein protein, but also has many new advantages, such as direct absorption, strong solubility, strong stability, high safety, etc., and thus has good development prospects.

[0044] Meanwhile, as a hydrolysate, the casein protein hydrolysate also has the advantages of low preparation cost and simple preparation method, which can overcome the problems of high synthesis cost or difficult purification and separation of active peptide segments, and thus is more easily applied to industrial large-scale production.

[0045] It has been verified through experiments that the casein hydrolysate provided by the present application 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] Further, the content of the peptide segment FAWPQYLK is ≥1.50% based on the mass of the casein hydrolysate; the content of the peptide segment LPW is ≥0.05% based on the mass of the casein hydrolysate; the content of the peptide segment MPLW is ≥0.05% based on the mass of the casein hydrolysate; and the content of the peptide segment PLW is ≥0.05% based on the mass of the casein hydrolysate.

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

[0048] In the above technical solution, by regulating the degree of hydrolysis of the casein hydrolysate, the enrichment of the active peptide segment can be promoted, thereby improving the anti-inflammatory effect of the casein hydrolysate; specifically, the degree of hydrolysis of the casein hydrolysate can be 10%-15%. In addition, by regulating the molecular weight of the casein hydrolysate, the casein hydrolysate can have the advantages of small molecular weight and easy absorption, and the enrichment of the active peptide segment can be promoted, thereby improving the anti-inflammatory effect of the casein hydrolysate; specifically, the mass content of the peptide with a molecular weight ≤10000 Da in the casein hydrolysate is ≥80%; the mass content of the peptide with a molecular weight ≤5000 Da in the casein hydrolysate is ≥55%; and the mass content of the peptide with a molecular weight <1000 Da in the casein hydrolysate is ≥30%. It can be understood that when at least one of the degree of hydrolysis and the molecular weight distribution of the casein hydrolysate of the present application 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 subjecting a casein raw material to enzymatic treatment, wherein the enzymatic treatment comprises using trypsin and streptokinase for enzymolysis.

[0050] Casein is the main protein in the milk of mammals (such as cows, sheep and humans). The present application can prepare casein hydrolysate by using casein as a raw material and subjecting it to enzymolysis, so as to promote the conversion of macromolecular casein into small molecular casein hydrolysate which can be directly absorbed and utilized by the body, has a definite characteristic structure and has biological activity, so as to improve the biological activity of casein, and at the same time, endow it with higher added value and more extensive use scenarios.

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

[0052] The present application realizes enzymatic treatment by using trypsin and streptomyces protease. The trypsin is a serine protease extracted from the pancreas of cattle, sheep and pigs, and is also an endopeptidase that can break the peptide bond formed by the carboxyl group of lysine or arginine. It has strong specificity and plays a key role in determining the amino acid arrangement of proteins. Streptomyces protease is an enzyme preparation purified and compounded by Aspergillus oryzae strain fermentation. It can form a unique flavor in the enzymatic product and reduce the bitterness caused by enzymatic hydrolysis. In the present application, by limiting the types of enzyme preparation to meet the above range, the active peptides can be further enriched to improve the anti-inflammatory efficacy of the casein protein hydrolysate.

[0053] The third aspect of the present application provides a preparation method of casein protein hydrolysate, comprising the following steps:

[0054] The casein protein raw material is subjected to enzymatic treatment to obtain casein protein hydrolysate.

[0055] The enzymatic treatment includes using trypsin and streptomyces protease for enzymatic hydrolysis.

[0056] The present application has conducted a lot of research and exploration on how to promote the presence of the expected active peptide segments FAWPQYLK, LPW, MPLW or PLW in the enzymatic product of the casein protein raw material, and it is proved that the selection of enzyme preparation has a key impact on the result. The use of trypsin and streptomyces protease for enzymatic treatment of casein protein raw material can obtain casein protein hydrolysate with expected active peptide segments. The above preparation method by limiting enzyme preparation can simply and quickly obtain casein protein hydrolysate containing expected active peptides, which is convenient for large-scale production.

[0057] During the enzymatic treatment, trypsin and streptomyces protease can be used for enzymatic treatment at the same time. In order to increase the contact area of enzyme preparation and casein protein raw material, and ensure the uniform mixing of enzyme preparation and casein protein raw material, and improve the efficiency of enzymatic hydrolysis, the trypsin and streptomyces protease can be mixed in a small amount of pure water to obtain an enzyme mixture. Then the enzyme mixture is added to the casein protein for enzymatic treatment.

[0058] After the enzymatic treatment is completed, enzyme inactivation treatment can be performed to make the enzyme preparation lose catalytic activity. The present application does not limit the way of enzyme inactivation, and the enzyme inactivation can be performed by conventional enzyme inactivation means in the art, for example, enzyme inactivation treatment can be performed at 80℃ for 30 min.

[0059] After the enzyme inactivation treatment, the temperature can be lowered to 30-40°C, and then concentrated and dried. The concentration treatment can further enrich the active peptides, and the drying treatment can obtain the solid form of the casein hydrolysate, which is convenient for storage and transportation. The present application does not limit the concentration treatment and drying treatment methods, which 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-drying or spray drying.

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

[0061] Further, the enzymatic effect of the enzyme preparation can be better exerted by adjusting the enzymatic conditions. Specifically, the enzymatic conditions include: the enzymatic temperature is 40-55°C, the enzymatic time is 2.0-3.5 h, or the enzymatic pH value is 7.5-8.2. In the present application, the casein raw material can be first adjusted to the optimal enzymatic pH environment of the enzyme mixture, i.e. the pH value is 7.5-8.2, to obtain an enzymatic stock solution that is beneficial to the enzymatic reaction of the enzyme mixture. Subsequently, under the suitable enzymatic temperature of the enzyme mixture, i.e. 40-55°C, the enzyme mixture composed of two kinds of enzyme preparations is added to the enzymatic stock solution, and then the enzymatic reaction is carried out for 2.0-3.5 h, so that the two kinds of enzyme preparations can fully act on the proteins in the enzymatic stock solution.

[0062] To better enrich the active peptide fragments, in the present application, the casein and water can be first mixed according to a mass ratio of 1:(8-12) to obtain a casein solution, and then the casein solution is used as the casein raw material for enzymatic reaction. Since the casein solution has a certain flowability, its surface area is larger than that of the casein powder, and thus it is more conducive to promoting the subsequent enzymatic reaction. If too little water is added, the flowability of the casein solution will be poor, which is not conducive to the action of the enzyme preparation, and is easy to cause the enzymatic efficiency to decrease; if too much water is added, the reaction volume will be too large during the enzymatic treatment, and thus 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 application, the preparation method of the casein protein hydrolysate is as follows: casein protein powder and water are mixed according to a volume ratio of 1: (8-12) to obtain a casein protein 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 protein powder) and streptomyces proteinase 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 protein powder) are added to the casein protein solution for enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 40-55℃, the enzymatic hydrolysis time is 2.0-3.5 h, and the enzymatic hydrolysis pH value is 7.5-8.2, to obtain the casein protein hydrolysate.

[0064] In an embodiment, the casein protein and water can be mixed according to a mass ratio of 1:10, trypsin with an enzyme activity of 200000 U / g and an addition amount of 0.25wt% (based on the mass of the casein protein) and streptomyces proteinase with an enzyme activity of 200000 U / g and an addition amount of 0.12wt% (based on the mass of the casein protein) are added thereto for enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 45℃, the enzymatic hydrolysis pH value is 7.5, the enzymatic hydrolysis time is 2.0 h, to obtain an enzymatic hydrolysate, and then the enzymatic hydrolysate is subjected to enzyme inactivation treatment, evaporation concentration treatment and drying treatment, to obtain the casein protein hydrolysate.

[0065] In an embodiment, the casein protein and water can be mixed according to a mass ratio of 1:12, trypsin with an enzyme activity of 200000 U / g and an addition amount of 0.17wt% (based on the mass of the casein protein) and streptomyces proteinase with an enzyme activity of 200000 U / g and an addition amount of 0.17wt% (based on the mass of the casein protein) are added thereto for enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 45℃, the enzymatic hydrolysis pH value is 7.5, the enzymatic hydrolysis time is 2.5 h, to obtain an enzymatic hydrolysate, and then the enzymatic hydrolysate is subjected to enzyme inactivation treatment, evaporation concentration treatment and drying treatment, to obtain the casein protein hydrolysate.

[0066] In an embodiment, the casein protein and water can be mixed according to a mass ratio of 1:8, trypsin with an enzyme activity of 200000 U / g and an addition amount of 0.35wt% (based on the mass of the casein protein) and streptomyces proteinase with an enzyme activity of 200000 U / g and an addition amount of 0.05wt% (based on the mass of the casein protein) are added thereto for enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 45℃, the enzymatic hydrolysis pH value is 8.2, the enzymatic hydrolysis time is 3.0 h, to obtain an enzymatic hydrolysate, and then the enzymatic hydrolysate is subjected to enzyme inactivation treatment, evaporation concentration treatment and drying treatment, to obtain the casein protein hydrolysate.

[0067] In an embodiment, the casein protein and water can be mixed at a mass ratio of 1:10, trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.10 wt% (based on the mass of the casein protein) and streptomyces proteinase with an enzyme activity of 200,000 U / g and an addition amount of 0.32 wt% (based on the mass of the casein protein) are added to the mixture for enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH value is 7.5, the enzymatic hydrolysis time is 3.5 h, and a hydrolysis liquid is obtained, and then enzyme inactivation treatment, evaporation concentration treatment and drying treatment are performed to obtain the casein protein hydrolysate.

[0068] In the present application, the casein protein and water can be mixed at a mass ratio of 1:(8-12), trypsin with an enzyme activity of 180-220,000 U / g and an addition amount of 0.10 wt%-0.35 wt% (based on the mass of the casein protein) and streptomyces proteinase with an enzyme activity of 180-220,000 U / g and an addition amount of 0.05 wt%-0.32 wt% (based on the mass of the casein protein) are added to the mixture for enzymatic hydrolysis treatment, 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 a hydrolysis liquid is obtained, and then enzyme inactivation treatment, evaporation concentration treatment and drying treatment are performed to obtain the casein protein hydrolysate.

[0069] The present application provides the use of the casein protein hydrolysate or the casein protein hydrolysate prepared by the preparation method in the preparation of related products with anti-inflammatory effect.

[0070] It can be understood that the related products include but are not limited to skin care products, cosmetics and drugs. A large amount of research data prove that the casein protein hydrolysate of the present application including the peptide segment FAWPQYLK, the peptide segment LPW, the peptide segment MPLW and the peptide segment PLW can reduce the inflammatory response of cells by reducing the release of pro-inflammatory cytokines and increasing the release of anti-inflammatory cytokines, thereby achieving the anti-inflammatory effect. Therefore, the casein protein hydrolysate including the active peptide segment and the casein protein hydrolysate prepared by the preparation method can not only be applied to skin care products, cosmetics in the conventional sense, such as anti-inflammatory skin care products or anti-inflammatory cosmetics, but also can be used for the preparation of drugs with anti-inflammatory effect, thereby widening the application range of the casein protein hydrolysate and providing a new raw material for related products with anti-inflammatory effect.

[0071] In addition, it has been verified through experiments that the casein protein hydrolysate and the casein protein hydrolysate prepared by the preparation method can be applied to relieve the inflammatory response of THP-1 M1 macrophages or be applied to the preparation of drugs related to the inflammatory response of THP-1 M1 macrophages.

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

[0073] Hereinafter, the technical solutions of the present application will be further explained and described in combination with specific examples. In the following examples, the experimental methods not specified in terms of specific conditions are generally performed according to conventional conditions or according to the conditions suggested by the manufacturers. The reagents used, if not specifically stated, are commercially available or can be obtained from public channels.

[0074] Example 1: Preparation of milk casein hydrolysate

[0075] (1) The milk casein powder and pure water were mixed according to a mass ratio of 1:10 to obtain a milk casein solution. The trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.25 wt% (based on the mass of the milk casein powder) and the streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.12 wt% (based on the mass of the milk casein powder) were uniformly mixed in 10 mL of pure water to obtain an enzyme mixture. The enzyme mixture was added to the milk casein solution for enzymatic hydrolysis treatment to obtain an enzymatic hydrolysate. In the enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature was 45°C, the enzymatic hydrolysis pH value was 7.5, and the enzymatic hydrolysis time was 2.0 h. After the enzymatic hydrolysis treatment, the enzymatic hydrolysate was subjected to enzyme inactivation treatment at 80°C for 30 min. After the enzymatic hydrolysate after the enzyme inactivation treatment was cooled to 30-40°C, it was subjected to evaporation concentration treatment and vacuum freeze-drying treatment to obtain milk casein hydrolysate powder 1.

[0076] (2) The cheese protein powder and pure water were mixed according to a mass ratio of 1:12 to obtain a cheese protein solution. The trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.17wt% (based on the mass of the cheese protein powder) and the streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.17wt% (based on the mass of the cheese protein powder) were uniformly mixed in 10 mL of pure water to obtain an enzyme mixture. The enzyme mixture was added to the cheese protein solution for enzymolysis treatment to obtain an enzymolysis solution. In the enzymolysis treatment, the enzymolysis temperature was 45°C, the enzymolysis pH value was 7.5, and the enzymolysis time was 2.5 h. After the enzymolysis treatment, the enzymolysis solution was subjected to enzyme inactivation treatment at 80°C for 30 min. After the enzymolysis solution after the enzyme inactivation treatment was cooled to 30-40°C, evaporation concentration treatment and vacuum freeze-drying treatment were performed to obtain cheese protein hydrolysate powder 2.

[0077] (3) The cheese protein powder and pure water were mixed according to a mass ratio of 1:8 to obtain a cheese protein solution. The trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.35wt% (based on the mass of the cheese protein powder) and the streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.05wt% (based on the mass of the cheese protein powder) were uniformly mixed in 10 mL of pure water to obtain an enzyme mixture. The enzyme mixture was added to the cheese protein solution for enzymolysis treatment to obtain an enzymolysis solution. In the enzymolysis treatment, the enzymolysis temperature was 45°C, the enzymolysis pH value was 8.2, and the enzymolysis time was 3.0 h. After the enzymolysis treatment, the enzymolysis solution was subjected to enzyme inactivation treatment at 80°C for 30 min. After the enzymolysis solution after the enzyme inactivation treatment was cooled to 30-40°C, evaporation concentration treatment and vacuum freeze-drying treatment were performed to obtain cheese protein hydrolysate powder 3.

[0078] (4) The cheese protein powder and pure water were mixed according to a mass ratio of 1:10 to obtain a cheese protein solution. The trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.10wt% (based on the mass of the cheese protein powder) and the streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.32wt% (based on the mass of the cheese protein powder) were uniformly mixed in 10 mL of pure water to obtain an enzyme mixture. The enzyme mixture was added to the cheese protein solution for enzymolysis treatment to obtain an enzymolysis solution. In the enzymolysis treatment, the enzymolysis temperature was 45°C, the enzymolysis pH value was 7.5, and the enzymolysis time was 3.5 h. After the enzymolysis treatment, the enzymolysis solution was subjected to enzyme inactivation treatment at 80°C for 30 min. After the enzymolysis solution after the enzyme inactivation treatment was cooled to 30-40°C, evaporation concentration treatment and vacuum freeze-drying treatment were performed to obtain cheese protein hydrolysate powder 4.

[0079] Example 2: Detection of cheese protein hydrolysate

[0080] (1) Detection of the degree of hydrolysis of cheese protein hydrolysate

[0081] The degree of hydrolysis of the casein protein hydrolysate powders 1-4 in Example 1 was determined by OPA reaction. Since one free amine group is released for each peptide bond hydrolyzed, the free amine group reacts with OPA (o-phenylenediamine) to form a yellow complex. Therefore, the degree of hydrolysis can be characterized by measuring the absorbance at 340 nm by spectrophotometer. The calculation formula of the degree of hydrolysis is as follows:

[0082]

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

[0084] After three parallel experiments, the degrees of hydrolysis of casein protein hydrolysate 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) Detection of molecular weight distribution of casein protein hydrolysate

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

[0087] Table 1

[0088]

[0089] As can be seen from Table 1, the distribution of molecular weight of casein protein hydrolysate powders 1-4 is similar, and the mass content of peptides with molecular weight ≤10000 Da is ≥80%, the mass content of peptides with molecular weight ≤5000 Da is ≥55%, and the mass content of small molecular peptides and amino acids with molecular weight <1000 Da is ≥30%.

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

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

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

[0093] The detection samples 1-4 were subjected to LC-MS / MS detection respectively, and the capillary liquid chromatography conditions were as follows: analysis column: 150 μm i.d.×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 of each component: 66 min. The 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 spectrometry raw files obtained after LC-MS / MS detection were searched against the Uniprot protein database using Maxquant (1.6.2.10) to obtain the amino acid sequences of the characteristic peptides in the casein hydrolysate powders 1-4 and their content ranges.

[0095] (2) Screening of bioactive peptides

[0096] The characteristic peptides common to the casein hydrolysate powders 1-4 were subjected to prediction of potential biological activities using the online prediction tool Peptide Ranker. The score of the peptides in the Peptide Ranker software was from 0 to 1, and the higher the score, the higher the possibility of biological activity of the peptide. The score was based on the fact that the specific structural features and amino acid sequences of the bioactive peptides promoted the specific biological activities of the bioactive peptides. Polypeptides with a score >0.8 were screened as bioactive peptides, and a total of 54 polypeptides were obtained, as shown in Table 2.

[0097] Table 2

[0098]

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

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

[0101] Table 3

[0102]

[0103] Example 4: Function verification of milk casein hydrolysate and anti-inflammatory active peptide segments

[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 secretes various cytokines to regulate inflammation to stimulate, recruit, and expand immune cells, leading to the occurrence of inflammatory reactions. Inflammation is a defense mechanism against harmful substances invading life, allowing damaged tissues to repair and heal. However, if the inflammatory response is too strong or lasts too long, it can also cause a series of damage to the body, and in severe cases, even endanger life.

[0105] Cytokines (Cytokine, CK) are a class of small molecular proteins with broad biological activity, such as interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α); 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.) upon 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 a biological effect of inhibiting inflammatory responses and helps to resolve inflammation and recover from the acute phase of autoimmune diseases. Pro-inflammatory cytokines include IL-1β, IL-2, IL-6, and TNF-α, which can signal through type I cytokine receptors (CCR1) to regulate cell-mediated immune responses, promote the growth, activation, differentiation, and homing of immune cells to infection sites, and help the occurrence and spread of autoimmune inflammation.

[0106] Therefore, to verify the anti-inflammatory effect of the cheese protein hydrolysate and the anti-inflammatory active peptide segment, the content of pro-inflammatory cytokines and anti-inflammatory cytokines released by the macrophage-like cell inflammation model under the treatment of the cheese protein hydrolysate and the anti-inflammatory active peptide segment is detected to characterize the anti-inflammatory effect of the cheese protein hydrolysate and the anti-inflammatory active peptide segment.

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

[0108] The routine culture and subculture operation of human mononuclear cells (THP-1) are performed using the basic culture medium under the condition of 37°C and 5% CO2, wherein the basic culture medium is RPMI-1640 culture medium containing 10% fetal bovine serum, 0.05 mM β-mercaptoethanol and 1% penicillin-streptomycin solution, and the THP-1 cells, the basic culture medium and the components thereof are purchased from Nanjing Sunbioga Biotechnology Co., Ltd. Before the start of the enzyme-linked immunosorbent assay, the THP-1 cells are inoculated in a 12-well plate at a density of 10 4 cells / cm 2 2, and 100 ng / mL of phorbol ester (PMA) is added for induction treatment for 24-48 h to induce the THP-1 cells into macrophage-like cells.

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

[0110] The macrophage-like cells in (1) are added with 20 ng / mL of lipopolysaccharide (LPS) for stimulation treatment for 6 h, and then the basic culture medium is replaced with serum-free culture medium without 10% fetal bovine serum for further culture for 24 h for starvation treatment to assimilate the cell state, thereby constructing a macrophage-like cell inflammation model to simulate the inflammatory response of macrophages.

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

[0112] The old culture medium of the macrophage-like cells in (1) is removed, new basic culture medium is added and cultured for 24 h, and the culture supernatant is collected. The culture supernatant is set as the Blank group (blank control group). The old culture medium of the macrophage-like cell inflammation model in (2) is removed, new basic culture medium is added and cultured for 24 h, and the culture supernatant is collected. The culture supernatant is set as the LPS group (experimental control group). The old culture medium of the macrophage-like cell inflammation model in (2) is removed, test culture medium is added and cultured for 24 h, and the culture supernatant is collected. The culture supernatant is set as the peptide segment FAWPQYLK group, the peptide segment LPW group, the peptide segment MPLW group, the peptide segment PLW group and the cheese protein hydrolysate group (experimental group) according to the test components of the test culture medium. The test culture medium is the basic culture medium containing different final concentrations of test components, and the different final concentrations of test components are shown in Table 4.

[0113] Table 4

[0114]

[0115] The culture supernatants of each group were detected using an ELISA kit produced by Wuhan Elabscience Biotechnology Co., Ltd. to obtain the contents of cytokines IL-1β, IL-2, IL-6, IL-10 and TNF-α in each group, which can be seen in detail in Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Figure 9 . The detection operation was performed according to the instructions of the ELISA kit.

[0116] Figure 1 Figure for the detection results of the cytokine content of the peptide segment 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 segment 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 the peptide segment FAWPQYLK, the content of cytokine IL-10 was higher than that in the LPS group; after treatment with medium concentration of the peptide segment FAWPQYLK, the content of cytokine IL-10 was not statistically different from that in the LPS group; after treatment with high concentration of the peptide segment FAWPQYLK, the content of cytokine IL-10 was lower than that in the LPS group; the content of IL-10 was concentration-dependent, and the lower the treatment concentration of the peptide segment FAWPQYLK, the higher the content of IL-10. Figure 1 The results showed that the peptide segment FAWPQYLK could reduce the inflammatory response of cells by significantly reducing the contents of cytokines IL-1β, IL-2, IL-6 and TNF-α, thereby achieving anti-inflammatory efficacy.

[0117] Figure 2 Figure for the detection results of the cytokine content of the peptide segment LPW group. Figure 2The results showed that the levels 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 LPW peptide, the levels of cytokines IL-1β, IL-6, and TNF-α were significantly lower than those in the LPS group. The levels of cytokines IL-1β, IL-6, and TNF-α showed a concentration-dependent effect; the higher the concentration of LPW peptide, the lower the levels of IL-1β, IL-6, and TNF-α. After treatment with low and medium concentrations of LPW peptide, there was no statistically significant difference in the level of IL-2 between the LPS and LPS groups. After treatment with high concentrations of LPW peptide, the level of IL-2 was lower than that in the LPS group. The level of IL-2 also showed a concentration-dependent effect; the higher the concentration of LPW peptide, the lower the level of IL-2. After treatment with low and medium concentrations of peptide LPW, the content of cytokine IL-10 was higher than that of the LPS group; after treatment with high concentrations of peptide LPW, the content of cytokine IL-10 was not statistically different from that of 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 could reduce cellular inflammatory response and achieve anti-inflammatory effects by significantly decreasing the levels of cytokines IL-1β, IL-6 and TNF-α and increasing the level of cytokine IL-10.

[0118] Figure 3 This is a graph showing the results of cytokine content detection in the MPLW peptide group. Figure 3 The results showed that the levels 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 MPLW, the levels of cytokines IL-1β, IL-2, IL-6, and TNF-α were significantly lower than those in the LPS group; the levels of cytokines IL-1β, IL-2, IL-6, and TNF-α did not exhibit typical concentration-dependent characteristics. After treatment with low, medium, and high concentrations of the peptide MPLW, the level of cytokines IL-10 was significantly higher than that in the LPS group; the level of IL-10 showed a concentration-dependent effect, with higher levels of IL-10 at lower MPLW treatment concentrations. Figure 3 The results showed that the peptide MPLW could reduce cellular inflammatory response and achieve anti-inflammatory effects by significantly decreasing the levels of cytokines IL-1β, IL-2, IL-6 and TNF-α, and increasing the level of cytokine IL-10.

[0119] Figure 4 Figure 4 is a graph showing the results of detecting the content of cytokines in the peptide PLW group. Figure 4 The results show that the contents of cytokines IL-1β, IL-2, IL-6, IL-10 and TNF-α in the LPS group are significantly higher than those in the Blank group, indicating that the macrophage inflammation model is successfully constructed by using LPS in this experiment. After being treated with low (L), medium (M) and high (H) concentrations of the peptide PLW, the contents of cytokines IL-1β, IL-2, IL-6 and TNF-α are significantly lower than those in the LPS group; the content of cytokine IL-2 has no concentration dependence; the contents of cytokines IL-1β, IL-6 and TNF-α have concentration dependence, and the higher the concentration of the peptide PLW, the lower the contents of cytokines IL-1β, IL-6 and TNF-α. After being treated with low concentration of the peptide PLW, the content of cytokine IL-10 is higher than that in the LPS group; after being treated with medium and high concentrations of the peptide PLW, the content of cytokine IL-10 has no statistical difference with that in the LPS group; the content of cytokine IL-10 has concentration dependence, and the lower the concentration of the peptide PLW, the higher the content of cytokine IL-10. Figure 4 The results show that the peptide PLW can reduce the inflammatory response of cells by significantly reducing the contents of cytokines IL-1β, IL-2, IL-6 and TNF-α, and achieve the anti-inflammatory effect.

[0120] Figure 5 Figure 6 is a graph showing the results of detecting the content of cytokine TNF-α in the milk casein hydrolysate group, Figure 6 Figure 7 is a graph showing the results of detecting the content of cytokine IL-10 in the milk casein hydrolysate group, Figure 7 Figure 8 is a graph showing the results of detecting the content of cytokine IL-6 in the milk casein hydrolysate group, Figure 8 Figure 9 is a graph showing the results of detecting the content of cytokine IL-2 in the milk casein hydrolysate group, Figure 9 Figure 10 is a graph showing the results of detecting the content of cytokine IL-1β in the milk casein hydrolysate group. Figures 5-9The results show that the contents of cytokines IL-1β, IL-2, IL-6, IL-10 and TNF-α in the LPS group are significantly higher than those in the Blank group, indicating that the macrophage inflammation model is successfully constructed by using LPS in this experiment. In addition, after being treated with low (0.5 mg / mL), medium (2.3 mg / mL) and high (4.1 mg / mL) concentrations of milk casein hydrolysate powder 1, the contents of cytokines IL-1β, IL-2 and IL-6 are significantly lower than those in the LPS group (p<0.001), indicating that milk 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 effect. After being treated with low (0.5 mg / mL), medium (2.3 mg / mL) and high (4.1 mg / mL) concentrations of milk casein hydrolysate powder 2, the contents of cytokines IL-1β, IL-2 and IL-6 are significantly lower than those in the LPS group, indicating that milk casein hydrolysate powder 2 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 effect. After being treated with low (0.5 mg / mL), medium (2.3 mg / mL) and high (4.1 mg / mL) concentrations of milk casein hydrolysate powder 3, the content of cytokine IL-1β is significantly lower than that in the LPS group, and at medium and high concentrations, the contents of cytokines IL-2 and IL-6 are significantly lower than those in the LPS group, indicating that milk casein hydrolysate powder 3 can also reduce the inflammatory response of cells by reducing the contents of cytokines IL-1β, IL-2 and IL-6, thereby achieving anti-inflammatory effect. After being treated with low (0.5 mg / mL), medium (2.3 mg / mL) and high (4.1 mg / mL) concentrations of milk casein hydrolysate powder 4, the contents of cytokines IL-1β, IL-2 and IL-6 are significantly lower than those in the LPS group, indicating that milk casein hydrolysate powder 4 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 effect.

[0121] In summary, the peptide segment FAWPQYLK, the peptide segment LPW, the peptide segment MPLW and the peptide segment PLW can reduce the inflammatory response of cells by reducing the contents of cytokines IL-1β, IL-2, IL-6 and TNF-α, and increasing the content of cytokine IL-10 at low concentration, thereby achieving anti-inflammatory effect. In addition, the milk casein hydrolysate containing the above four active peptide segments can also reduce the inflammatory response of cells by reducing the contents of cytokines IL-1β, IL-2 and IL-6, thereby achieving anti-inflammatory effect.

[0122] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and 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 application.

Claims

1. A method for the preparation of a milk casein hydrolysate, characterized in that, The method comprises the following steps: carrying out enzymatic hydrolysis on a casein protein raw material to obtain a casein protein hydrolysate; the enzymatic hydrolysis comprises using trypsin and streptomyces protease for enzymatic hydrolysis; the trypsin has an enzyme activity of 180000-220000 U / g; the trypsin is added in an amount of 0.10-0.35 wt% based on per gram of the casein protein raw material; the streptomyces protease has an enzyme activity of 180000-220000 U / g; the streptomyces protease is added in an amount of 0.05-0.32 wt% based on per gram of the casein protein raw material.

2. The production method according to claim 1, characterized by, the enzymatic hydrolysis is carried out under conditions of an enzymatic hydrolysis temperature of 40-55℃, an enzymatic hydrolysis time of 2.0-3.5 h, or an enzymatic hydrolysis pH value of 7.5-8.

2.

3. The production method according to claim 1 or 2, characterized by, the casein protein raw material is obtained by mixing casein protein and water in a volume ratio of 1: (8-12).

4. The milk casein hydrolysate obtainable by the process according to any one of claims 1 to 3, characterized in that, the casein protein hydrolysate comprises a peptide segment FAWPQYLK, a peptide segment LPW, a peptide segment MPLW and a peptide segment PLW; the content of the peptide segment FAWPQYLK is 1.97%-4.65% based on the mass of the casein protein hydrolysate; the content of the peptide segment LPW is 0.09%-0.12% based on the mass of the casein protein hydrolysate; the content of the peptide segment MPLW is 0.07%-0.08% based on the mass of the casein protein hydrolysate; the content of the peptide segment PLW is 0.09%-0.13% based on the mass of the casein protein hydrolysate; the degree of hydrolysis of the casein protein hydrolysate is 10%-15%; the mass content of peptides with a molecular weight of ≤10000 Da in the casein protein hydrolysate is ≥80%; the mass content of peptides with a molecular weight of ≤5000 Da in the casein protein hydrolysate is ≥55%; the mass content of peptides with a molecular weight of <1000 Da in the casein protein hydrolysate is ≥30%.

5. The casein protein hydrolysate prepared by the preparation method in any one of claims 1-3, and / or the casein protein hydrolysate in claim 4 is applied to preparation of a related product having anti-inflammatory efficacy.

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