A kind of goat milk casein enzymatic hydrolysate and preparation method and application thereof

Abstract

The present invention provides a goat casein protein hydrolysate, a preparation method and an application thereof. The goat casein protein hydrolysate provided by the present invention has an antioxidant effect, which is mainly achieved by regulating the types and contents of functional peptides therein. Among them, the functional peptides include peptide segments PWP, MPLW, PLW and LPW, which have biological activity, the potential to bind to and activate Kelch-like ECH-associated protein 1 (Keap1), and the ability to scavenge superoxide anions, and thus have an antioxidant effect.

Description

Technical Field

[0001] The invention relates to biotechnology, and in particular to a goat casein protein hydrolysate, a preparation method and an application thereof. Background Art

[0002] Oxidative stress is caused by an imbalance between pro-oxidants (free radicals) and the body's scavenging ability (antioxidants). Reactive oxygen species (ROS), as one of the body's oxidatively active molecules, are closely related to the occurrence of oxidative stress, and mainly include superoxide anions, hydroxyl radicals, peroxyl radicals, and hydrogen peroxide. ROS participates in the synthesis of human active substances, cell detoxification, immune function, etc. As an important second messenger, it participates in intercellular signal transduction and regulates gene expression to stabilize the intracellular environment. However, when ROS increases too much, it will cause damage to the body, often involving lipid peroxidation, proteins, nucleic acids and other substances, and ultimately cause oxidative stress in the body.

[0003] Currently, the commonly used antioxidants are all artificially synthesized. Although they have good effects and are cheap, their long-term safety cannot be guaranteed. Therefore, finding an antioxidant with good antioxidant effect and safety and non-toxicity has become a research hotspot. Summary of the invention

[0004] The invention provides a goat milk casein protein hydrolysate, which has the effect of promoting and restoring the oxidation / antioxidation balance of the body.

[0005] The invention provides a method for preparing the goat cheese protein hydrolysate, which can simply and quickly obtain goat cheese hydrolysate containing expected functional peptides, and is convenient for expanding production.

[0006] The present invention also provides a use of the goat casein enzymatic hydrolysate or the goat casein enzymatic hydrolysate prepared by the above-mentioned preparation method in the preparation of antioxidant related products.

[0007] The present invention provides a goat milk casein enzymatic hydrolysate, wherein the goat milk casein enzymatic hydrolysate comprises a peptide segment PWP, a peptide segment MPLW, a peptide segment PLW, and a peptide segment LPW;

[0008] Based on the mass of goat casein enzymatic hydrolysate, the content of peptide PWP is ≥0.08%, the content of peptide MPLW is ≥0.65%, the content of peptide PLW is ≥0.09%, and the content of peptide LPW is ≥0.37%.

[0009] The goat milk casein enzymatic hydrolysate as described above, wherein the degree of hydrolysis of the goat milk casein enzymatic hydrolysate is 10%-15%; and / or,

[0010] The mass content of peptides with a molecular weight of ≤3000 Da in goat casein enzymatic hydrolysate is greater than 90%, and the mass content of peptides with a molecular weight of <1000 Da is greater than 60%.

[0011] The goat milk casein enzymatic hydrolysate as described above, wherein the goat milk casein enzymatic hydrolysate is obtained by enzymatically hydrolyzing goat milk casein as a raw material;

[0012] The enzymatic treatment comprises using at least one of trypsin, alkaline protease, bromelain and flavor protease.

[0013] The present invention provides a method for preparing the above-mentioned goat casein enzymatic hydrolysate, comprising: enzymolyzing goat casein with a protease to obtain the goat casein enzymatic hydrolysate;

[0014] The protease includes at least one of trypsin, alkaline protease, bromelain and flavor protease.

[0015] The preparation method as described above, wherein, based on each gram of goat milk casein, the enzyme activity of trypsin is 4000 U, and the addition amount is 0.05-0.6wt%; the enzyme activity of alkaline protease is 200000 U, and the addition amount is 0.1-0.6wt%; the enzyme activity of bromelain is 50000 U, and the addition amount is 0.1-0.6wt%; the enzyme activity of flavor protease is 20000 U, and the addition amount is 0.1-0.6wt%.

[0016] The preparation method as described above, wherein the enzymatic hydrolysis temperature is 40-50°C, the time is 1.5-5.0 h, and the pH value is 7.0-9.0.

[0017] The preparation method as described above, wherein the protease comprises trypsin.

[0018] The present invention also provides a use of the goat casein enzymatic hydrolysate or the goat casein enzymatic hydrolysate prepared by the above-mentioned preparation method in the preparation of antioxidant related products.

[0019] The present invention provides a goat milk casein enzymatic hydrolysate, which has an antioxidant effect, which is mainly achieved by regulating the types and contents of functional peptides therein. Among them, the functional peptides include peptide segments PWP, peptide segments MPLW, peptide segments PLW and peptide segments LPW, which have biological activity, the potential to bind and activate Kelch-like ECH-associated protein 1 (Keap1), and the ability to scavenge superoxide anions. The nuclear factor E2-related factor 2 / Kelch-like ECH-associated protein 1-antioxidant response element (Nrf2 / Keap1-ARE) signaling pathway is one of the most important defense mechanisms of the body against oxidative stress. It regulates the transcription of multiple downstream cell protection genes, maintains intracellular redox balance and metabolism and protein homeostasis, and exerts biological functions such as anti-inflammatory, anti-cancer and anti-aging. Therefore, binding with Keap1 can activate the enzyme and then exert an antioxidant effect. The accumulation of superoxide anions in mitochondria can cause mitochondrial oxidative stress, thereby damaging lipids, DNA, proteins, etc. in mitochondria and inducing diseases. Therefore, reducing the content of superoxide anions is of great significance in promoting the restoration of the body's oxidation / antioxidation balance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the degree of hydrolysis of the goat milk casein enzymatic hydrolysate in Example 2;

[0021] Figure 2 is the IC of goat milk casein enzymatic hydrolysate No. 1 in Example 3 50 Value result graph;

[0022] Figure 3 is the IC of goat milk casein enzymatic hydrolysate No. 2 in Example 3 50 Value result graph;

[0023] Figure 4 is the IC of goat milk casein enzymatic hydrolysate No. 3 in Example 3 50 Value result graph. DETAILED DESCRIPTION

[0024] 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.

[0025] The first aspect of the present invention provides a goat milk casein enzymatic hydrolysate, wherein the goat milk casein enzymatic hydrolysate comprises a peptide segment PWP, a peptide segment MPLW, a peptide segment PLW, and a peptide segment LPW;

[0026] Based on the mass of goat casein enzymatic hydrolysate, the content of peptide PWP is ≥0.08%, the content of peptide MPLW is ≥0.65%, the content of peptide PLW is ≥0.09%, and the content of peptide LPW is ≥0.37%.

[0027] Goat Milk Casein Hydrolysates (GMCH) refers to the enzymatic hydrolysate obtained by enzymatic hydrolysis of goat milk casein. At present, although functional peptides have the advantages of defined composition, high safety, low allergy, high activity and easy absorption when used alone, the separation and purification cost of natural functional peptides is extremely high, which limits their application in industrial large-scale production. Therefore, the present invention provides a low-cost goat milk casein hydrolysate, which includes peptide segments PWP, peptide segments MPLW, peptide segments PLW, and peptide segments LPW, and has an antioxidant effect.

[0028] It can be understood that the peptide segment PWP is a tripeptide segment (Pro-Trp-Pro) consisting of proline, tryptophan, and proline from the N-terminus to the C-terminus, the peptide segment MPLW is a tetrapeptide segment (Met-Pro-Leu-Trp) consisting of methionine, proline, leucine, and tryptophan from the N-terminus to the C-terminus, the peptide segment PLW is a tripeptide segment (Pro-Leu-Trp) consisting of proline, leucine, and tryptophan from the N-terminus to the C-terminus, and the peptide segment LPW is a tripeptide segment (Leu-Pro-Trp) consisting of leucine, proline, and tryptophan from the N-terminus to the C-terminus.

[0029] In addition, in the present invention, based on the quality of goat casein enzymatic hydrolysate, the contents of peptide segment PWP, peptide segment MPLW, peptide segment PLW and peptide segment LPW are limited to meet the above ranges, which can enable the goat casein enzymatic hydrolysate to show good antioxidant effects.

[0030] After long-term research, it was found that the goat milk casein hydrolysate of the present invention has an antioxidant effect, which is mainly achieved by regulating the types and contents of functional peptides therein. Peptide segments PWP, MPLW, PLW and LPW have biological activity and have the potential to bind to Kelch-like ECH-associated protein 1 (Keap1). The nuclear factor E2-related factor 2 / Kelch-like ECH-associated protein 1-antioxidant response element (Nrf2 / Keap1-ARE) signaling pathway is one of the most important defense mechanisms of the body against oxidative stress. It regulates the transcription of multiple downstream cell protection genes, maintains intracellular redox balance and metabolic and protein homeostasis, and exerts biological functions such as anti-inflammatory, anti-cancer and anti-aging. Therefore, binding to Keap1 can activate the enzyme and thus exert an antioxidant effect. At the same time, peptides PWP, MPLW, PLW and LPW also have strong superoxide anion scavenging ability. Since the accumulation of superoxide anions in mitochondria can cause mitochondrial oxidative stress, thereby damaging lipids, DNA, proteins, etc. in mitochondria and inducing diseases, reducing the content of superoxide anions is of great significance in promoting the restoration of the body's oxidation / antioxidation balance.

[0031] Furthermore, the hydrolysis degree of goat casein protein hydrolysate is 10%-15%. By regulating the hydrolysis degree of goat casein protein to keep it within a suitable range, it is beneficial to promote the enrichment of functional peptides. In addition, the mass content of peptides with a molecular weight of ≤3000 Da in goat casein protein hydrolysate is greater than 90%, and the mass content of peptides with a molecular weight of <1000 Da is greater than 60%. By regulating the molecular weight distribution of goat casein protein hydrolysate to meet the above range, the goat casein protein hydrolysate can have the advantages of small molecular weight and easy absorption.

[0032] In a specific embodiment, the goat casein enzymatic hydrolysate is obtained by enzymatically hydrolyzing goat casein as a raw material, wherein the enzymatic hydrolysis includes using at least one of trypsin, alkaline protease, bromelain, and flavor protease.

[0033] Trypsin is a serine protease extracted from the pancreas of cattle, sheep and pigs. It is also a peptide endonuclease that can break the peptide bonds 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. Alkaline protease is an enzyme that can hydrolyze protein peptide bonds under alkaline conditions, and its optimal pH range is 9-11. Bromelain refers to a sulfhydryl protease extracted from pineapple juice, pineapple peel, etc., which preferentially hydrolyzes the peptide chains on the carboxyl side of basic amino acids or aromatic amino acids, selectively hydrolyzes fibrin, and can decompose muscle fibers. It has different optimal pH ranges for different proteins. Flavor protease, also known as flavor enzyme, is an enzyme preparation obtained by fermentation, purification and compounding of Aspergillus oryzae strains, which can make the enzymolysis product form a unique flavor and reduce the bitterness caused by enzymolysis. In the present invention, by enzymolysis of goat milk casein, while limiting the type of enzyme preparation to meet the above range, functional peptides can be further enriched to improve the antioxidant effect of goat milk casein hydrolysate.

[0034] The third aspect of the present invention provides a method for preparing a goat casein enzymatic hydrolysate, comprising: enzymatically hydrolyzing goat casein with a protease to obtain a goat casein enzymatic hydrolysate, wherein the protease comprises at least one of trypsin, alkaline protease, bromelain, and flavor protease.

[0035] A lot of research and exploration has been conducted on how to make the enzymatic hydrolyzate of goat milk casein contain the expected functional peptides, that is, contain peptide segments PWP, MPLW, PLW and LPW, and their content meets the above requirements. It has been proved that the selection of protease has a key influence on the result. In the research process, it was unexpectedly found that the enzymatic hydrolysis of goat milk casein using at least one of trypsin, alkaline protease, bromelain and flavor protease can obtain a goat milk casein hydrolyzate including peptide segments PWP, MPLW, PLW and LPW, and the peptide content meets the above requirements. By limiting the preparation method of the protease, goat milk cheese hydrolyzate containing the expected functional peptides can be obtained simply and quickly, which is convenient for expanding production.

[0036] In the present invention, if goat casein is in solid form, goat casein can be first mixed with water to obtain a goat casein solution, wherein the mass ratio of goat casein to water can be 1: (8-12), for example, 1 g of goat casein can be mixed with 8-12 g of water to prepare a goat casein solution. The goat casein solution has fluidity and a larger surface area than that of goat casein, which is more conducive to its full contact with the protease. If too little water is added, the fluidity of the goat casein solution will be poor, which is not conducive to its contact with the protease and is likely to reduce the enzymolysis efficiency; if too much water is added, the reaction volume of the enzymolysis reaction will be too large, which is also likely to reduce the enzymolysis efficiency, and the subsequent processing process will also be more difficult.

[0037] In addition, after the enzymatic hydrolysis is completed, an enzyme inactivation treatment can be performed to inactivate the catalytic activity of the protease. The present invention does not limit the enzyme inactivation method, and the enzyme inactivation can be performed by conventional enzyme inactivation methods in the art, such as heating to 90-100° C. and maintaining for 15-30 min.

[0038] After the enzyme inactivation treatment, the temperature can be lowered to 30-40°C and then filtered, concentrated and spray-dried to further enrich the functional peptides and obtain a solid form of goat casein enzymatic hydrolysate for easy storage and transportation. The present invention does not limit the methods of filtering, concentrating and spray drying, and can be carried out by conventional technical means in the art. For example, filtration can use ultrafiltration or nanofiltration; concentration can be reduced pressure concentration at 65°C; spray drying can set the inlet temperature to 180°C and the outlet temperature to 90°C.

[0039] In a specific embodiment, based on per gram of goat milk casein, the enzyme activity of trypsin is 4000 U, and the addition amount is 0.05-0.6wt%; the enzyme activity of alkaline protease is 200000 U, and the addition amount is 0.1-0.6wt%; the enzyme activity of bromelain is 50000 U, and the addition amount is 0.1-0.6wt%; the enzyme activity of flavor protease is 20000 U, and the addition amount is 0.1-0.6wt%. When the enzyme activity and addition amount of protease meet the above range, it is helpful to promote the enzymatic hydrolysis reaction of goat milk casein and protease better, and increase the content of functional peptides in goat milk casein hydrolysate.

[0040] Furthermore, when the enzymatic hydrolysis temperature is 40-50°C, the time is 1.5-5.0 h, and the pH value is 7.0-9.0, the enzymatic hydrolysis effect of the protease can be better exerted.

[0041] Specifically, the goat milk casein solution can be adjusted to the optimal enzymolysis pH environment of the protease, that is, the pH value is 7.0-9.0, to obtain an enzymolysis stock solution that is conducive to the enzymolysis of the protease. Subsequently, at the appropriate enzymolysis temperature of the protease, that is, 40-50 ° C, after adding the protease to the enzymolysis stock solution, the enzymolysis is performed for 1.5-5.0 h, so that the protease fully acts on the protein in the enzymolysis stock solution. Specifically, 3 mol / L of food-grade sodium hydroxide solution can be added to the goat milk casein solution, the temperature is raised to 40-50 ° C and the pH is adjusted to 7.0-9.0. In addition, in order to make the reaction more sufficient, stirring can be started when the food-grade sodium hydroxide solution is added, and the stirring speed can be 100 rpm.

[0042] It is understandable that when the protease is selected from different types, the appropriate enzymolysis temperature, enzymolysis time and enzymolysis pH value can be selected within the above range according to the type of enzyme.

[0043] In aspects of the present invention, the protease comprises trypsin.

[0044] After a large number of experiments, it was found that when goat casein is enzymatically hydrolyzed with protease to obtain goat casein enzymatic hydrolysate, if the protease includes trypsin, the antioxidant effect of the goat casein enzymatic hydrolysate can be significantly improved.

[0045] In some embodiments of the present invention, the protease is only trypsin, or the protease is at least one of trypsin, alkaline protease, bromelain, and flavor protease and at least includes trypsin, and the clearance rate of the obtained goat casein enzymatic hydrolysate in the superoxide anion scavenging experiment is greater than 20%; and the protease is at least one of alkaline protease, bromelain, and flavor protease, and the inhibition rate of the obtained goat casein enzymatic hydrolysate in the superoxide anion scavenging experiment is 5%-15%.

[0046] In addition, it was found in the experiment that when the protease is only trypsin, the amount of trypsin added can be 0.1-0.5% of the mass of the goat casein enzymatic hydrolysate; when the protease is at least one of trypsin, alkaline protease, bromelain, and flavor protease and at least includes trypsin, trypsin can be added first for the first enzymolysis, and then at least one of alkaline protease, bromelain, and flavor protease can be added for the second enzymolysis, and the amount of trypsin added can be 0.05-0.3% of the mass of the goat casein enzymatic hydrolysate, and the amount of alkaline protease, bromelain, and flavor protease added can be 0.1-0.5% of the mass of the goat casein enzymatic hydrolysate.

[0047] A fourth aspect of the present invention provides a use of the above-mentioned goat casein enzymatic hydrolysate or the goat casein enzymatic hydrolysate prepared by the above-mentioned preparation method in the preparation of antioxidant-related products.

[0048] It is understandable that antioxidant-related products include but are not limited to health products, medicines, and antioxidant detection products. A large amount of research data proves that the goat casein protein hydrolysate provided by the present invention, including peptide segments PWP, MPLW, PLW, and LPW, and the peptide content meets the above requirements, has a significant antioxidant effect, and can be applied not only to health products in the conventional sense, such as antioxidant health products, but also to the preparation of antioxidant drugs, etc., thereby broadening the scope of application of goat casein protein hydrolysate and providing new raw materials for antioxidant products.

[0049] The technical solution of the present application will be further explained below in conjunction with specific embodiments. The experimental methods in the following embodiments 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.

[0050] Example 1: Preparation of goat milk casein enzyme hydrolysate

[0051] (1) 5 kg of goat milk casein raw material and 50 kg of pure water were mixed, and 3 mol / L food-grade sodium hydroxide solution was added at a stirring speed of 100 rpm. The temperature was raised to 40°C and the pH was adjusted to 8.0. Then, trypsin with an enzyme activity of 4000 U / g and a mass of 25 g was added thereto for 2.5 h of enzymolysis. During the period, stirring was maintained and sodium hydroxide solution was continuously added to maintain the pH value of the solution at 7.9-8.1. After the enzymolysis was completed, the temperature was immediately raised to 90°C and kept warm for 20 min to inactivate the enzyme. After the temperature dropped to 50°C, the mixture was filtered, concentrated at 65°C, and spray-dried to obtain goat milk casein enzymolysate No. 1.

[0052] (2) 10 kg of goat milk casein raw material and 120 kg of pure water were mixed, and 3 mol / L food-grade sodium hydroxide solution was added at a stirring speed of 100 rpm. The temperature was raised to 50°C and the pH was adjusted to 8.5. Then, trypsin with an enzyme activity of 4000 U / g and a mass of 20 g was added thereto for the first enzymolysis for 1.5 h. During the period, stirring was maintained and sodium hydroxide solution was continuously added to maintain the pH value of the solution at 8.4-8.6. After the first enzymolysis was completed, alkaline protease with an enzyme activity of 200000 U / g and a mass of 50 g was added thereto and the pH was adjusted to 9.0 for the second enzymolysis for 2.0 h. During the period, stirring was maintained and sodium hydroxide solution was continuously added to maintain the pH value of the solution. After the second enzymolysis was completed, the temperature was immediately raised to 90°C and kept warm for 30 min to inactivate the enzyme. After the temperature dropped to 40°C, the mixture was filtered, concentrated at 65°C, and spray-dried to obtain goat milk casein hydrolysate No. 2.

[0053] (3) 20 kg of goat milk casein raw material and 160 kg of pure water were mixed, and 3 mol / L food-grade sodium hydroxide solution was added at a stirring speed of 100 rpm. The temperature was raised to 45°C and the pH was adjusted to 7.5. Then, trypsin with an enzyme activity of 4000 U / g and a mass of 20 g was added thereto for a first enzymolysis of 2.0 h. During the period, stirring was maintained and sodium hydroxide solution was continuously added to maintain the pH value of the solution at 7.4-7.6. After the first enzymolysis was completed, bromelain with an enzyme activity of 50000 U / g and a mass of 30 g and flavor protease with an enzyme activity of 20000 U / g and a mass of 60 g were added thereto. The second enzymolysis was carried out for 3.0 h while stirring was maintained and the pH value was 7.4-7.6. After the second enzymolysis was completed, the temperature was immediately raised to 90°C and kept at this temperature for 30 min to inactivate the enzyme. After the temperature dropped to 50°C, the mixture was filtered, concentrated at 65°C, and spray-dried to obtain goat milk casein hydrolysate No. 3.

[0054] (4) 10 kg of goat milk casein raw material and 100 kg of pure water were mixed, and 3 mol / L food-grade sodium hydroxide solution was added at a stirring speed of 100 rpm. The temperature was raised to 50°C and the pH was adjusted to 8.5. Then, alkaline protease with an enzyme activity of 200,000 U / g and a mass of 60 g was added thereto for the first enzymolysis for 1.5 h. During the period, stirring was maintained and sodium hydroxide solution was continuously added to maintain the pH value of the solution at 8.4-8.6. After the first enzymolysis was completed, flavor protease with an enzyme activity of 20,000 U / g and a mass of 20 g was added thereto. The second enzymolysis was carried out for 3.5 h while stirring and the pH value was maintained at 8.4-8.6. After the second enzymolysis was completed, the temperature was immediately raised to 90°C and kept warm for 30 min to inactivate the enzyme. After the temperature dropped to 50°C, the mixture was filtered, concentrated at 65°C, and spray-dried to obtain goat milk casein hydrolysate No. 4.

[0055] Example 2: Detection of goat milk casein hydrolysate

[0056] (1) Hydrolysis degree detection of goat milk casein hydrolysate

[0057] The hydrolysis degree of the above-mentioned goat milk casein enzymatic hydrolysates No. 1-4 was determined by OPA reaction. Since each hydrolysis of a peptide bond will release a free amine group, and the free amine group reacts with OPA (o-phthalaldehyde) to form a yellow complex, the degree of hydrolysis can be characterized by measuring its absorbance at 340 nm with a spectrophotometer. The calculation formula of the hydrolysis degree is as follows: 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 goat milk casein enzymatic hydrolysate samples No. 1-4, mmol / L; V is the fixed volume of goat milk casein enzymatic hydrolysate samples No. 1-4, L; N is the dilution multiple of goat milk casein enzymatic hydrolysate samples No. 1-4; m is the mass of goat milk casein enzymatic hydrolysate samples No. 1-4, g; w is the protein content in goat milk casein enzymatic hydrolysate samples No. 1-4, %; β is a constant, casein is 0.383; α is a constant, casein is 1.039.

[0058] The specific degree of hydrolysis can be seen Figure 1 , where samples 1-4 correspond to goat milk casein hydrolysates 1-4, respectively. The hydrolysis degree of goat milk casein hydrolysate 1 is 10.13%, the hydrolysis degree of goat milk casein hydrolysate 2 is 11.15%, the hydrolysis degree of goat milk casein hydrolysate 3 is 12.33%, and the hydrolysis degree of goat milk casein hydrolysate 4 is 12.22%.

[0059] (2) Molecular weight distribution detection of goat milk casein hydrolysate

[0060] The molecular weight distribution of the above-mentioned goat casein enzymatic hydrolysates No. 1-4 was determined by referring to the GPC / UV detection method in Appendix A of the national standard GB 31645-2018, and the results can be seen in Table 1, where samples 1-4 correspond to goat casein enzymatic hydrolysates No. 1-4, respectively. As can be seen from Table 1, the molecular weight distribution of goat casein enzymatic hydrolysates No. 1-4 is similar, and the mass content of peptides with a molecular weight of ≤3000 Da is greater than 90%, and the mass content of small molecule peptides and amino acids with a molecular weight of <1000 Da is greater than 60%.

[0061] Table 1

[0062]

[0063] Example 3: Screening of antioxidant functional peptides

[0064] (1) LC-MS / MS mass spectrometry identification

[0065] The above-mentioned goat casein enzymatic hydrolysates No. 1-4 were dissolved in ddH2O to obtain a goat casein enzymatic hydrolysate solution, which was then transferred to a 10 kD ultrafiltration tube and centrifuged at 4°C and 12000 rcf for 10 min to collect polypeptide samples less than 10 kD, and dithiothreitol solution was added to the polypeptide sample to make its final concentration of 10 mmol / L, and reduced in a 56°C water bath for 1 h, and then iodoacetic acid solution was added to make its final concentration of 50 mmol / L. After reacting in the dark for 40 min, it was desalted using a self-filled desalting column, and the solvent was evaporated in a 45°C vacuum centrifugal concentrator to obtain a sample to be tested.

[0066] The samples to be tested 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.

[0067] The mass spectrometry raw files obtained after LC-MS / MS detection were used to search the Uniprot protein database using Maxquant (1.6.2.10) to obtain candidate bioactive peptides and their amino acid sequences.

[0068] (2) Bioactivity screening

[0069] The online prediction tool Peptide Ranker was used to predict the potential biological activity of candidate bioactive peptides. 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 bioactive peptides contribute to the specific biological activity of bioactive peptides. Peptides with scores > 0.8 were screened as bioactive peptides, a total of 70, as shown in Table 2.

[0070] Table 2

[0071]

[0072] (3) Molecular docking screening

[0073] In order to screen functional peptides with antioxidant activity, the above bioactive peptides were molecularly docked with Kelch-like ECH-associated protein 1 (Keap1) (PDBID: 2FLU). According to the binding energy score, the number of hydrogen bonds shown in the wiring diagram, and the degree of reorganization with the Keap1 binding site, 8 bioactive peptides were screened, and the binding energy scores of the bioactive peptides can be seen in Table 3. Since the nuclear factor E2-related factor 2 / Kelch-like ECH-associated protein 1-antioxidant response element (Nrf2 / Keap1-ARE) signaling pathway is one of the most important defense mechanisms of the body against oxidative stress, it regulates the transcription of multiple downstream cell protection genes, maintains intracellular redox balance and metabolic and protein homeostasis, and exerts biological functions such as anti-inflammatory, anti-cancer and anti-aging. Therefore, binding with Keap1 can activate the enzyme and thus exert an antioxidant effect.

[0074] Through the binding energy score and the content distribution of bioactive peptides in goat casein enzymatic hydrolysate, 8 candidate functional peptides were screened, namely peptide 8 (WPP), peptide 9 (PWP), peptide 15 (MPLW), peptide 17 (PLW), peptide 19 (RW), peptide 25 (LPW), peptide 26 (WY), and peptide 62 (AWPQYLK).

[0075] Table 3

[0076]

[0077] (4) Functional verification of candidate functional peptides

[0078] Under normal conditions, a certain concentration of reactive oxygen species participates in cell signal transduction and maintains intracellular homeostasis. However, under pathological conditions, the oxidation system and the antioxidant system are out of balance, the level of reactive oxygen species increases abnormally and accumulates excessively in the body, leading to oxidative stress. Mitochondria are the main subcellular structures of the intracellular reactive oxygen source. The accumulation of superoxide anions in mitochondria can cause mitochondrial oxidative stress, thereby damaging lipids, DNA, proteins, etc. in mitochondria, which is considered to be the cause of the disease. Therefore, it is of great significance to reduce the content of superoxide anions and restore the body's oxidation / antioxidation balance. The present invention screens functional peptides with antioxidant activity by detecting the superoxide anion scavenging ability of candidate functional peptides in vitro.

[0079] The above 8 candidate functional peptides were synthesized by Fmoc solid phase organic synthesis method, and the purity of the candidate functional peptides was >97%. The goat casein hydrolysates No. 1-4 obtained in Example 1, the 8 candidate functional peptides synthesized, and 0.1 mol / L Tris-HCl buffer with a pH value of 8.2 were dissolved and diluted to a final concentration of 1 mg / mL to obtain the sample to be tested. 0.1 mol / L luminol solution and 5.0 mmol / L carbonate buffer solution with a pH value of 10.2 were mixed in a volume ratio of 1:9 to prepare a luminol buffer. 100 μL of the sample to be tested, 100 μL, 1.0 mol / L pyrogallol solution and 800 μL luminol buffer solution were added to the measuring tube in sequence, and the solution in the measuring tube was mixed by flicking, and the luminescence intensity of the sample solution was measured by a BPCL ultra-weak luminescence measuring instrument, wherein the total volume of the solution in the measuring tube was 1 mL, the reaction time was 150 s, the luminescence intensity was detected every 0.1 s, and distilled water was used as a blank. The operation of setting up the blank control is similar to that of the sample group to be tested, except that pure water is used to replace the sample to be tested. The peak luminescence intensity is recorded, and the superoxide anion clearance rate can be calculated based on the measured luminescence intensity. The calculation formula is as follows: (Blank-Sample) / Blank×100%, where Blank is the luminescence intensity of the blank control, and Sample is the luminescence intensity of the sample to be tested. The specific results are shown in Table 4.

[0080] As shown in Table 4, among the candidate functional peptides, the inhibition rate of peptide 15 (MPLW) can reach 34.53%±4.17%, and the inhibition rate of peptide 25 (LPW) can reach 31.98%±1.49%, which has a strong inhibitory effect; peptide segment 9 (PWP) and peptide segment 17 (PLW) also have obvious inhibitory effects, and their inhibition rates are 20.05%±3.23% and 21.77%±2.10%, respectively. In the goat milk casein enzymatic hydrolysate, the inhibition rates of goat milk casein enzymatic hydrolysate No. 1-3 are all over 20%, and the inhibition rate of goat milk casein enzymatic hydrolysate No. 4 is slightly weaker, also reaching 10.25%±2.97%, indicating that the use of trypsin for enzymatic hydrolysis in the preparation of goat milk casein enzymatic hydrolysate has a more significant promoting effect on superoxide anion inhibition activity.

[0081] According to the inhibition rate results, goat milk casein enzymatic hydrolysates 1-3 were selected for concentration IC 50 The final concentrations of goat milk casein hydrolysates No. 1-3 were set to 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL and 2 mg / mL, respectively, to obtain a series of samples to be tested, and then the concentration IC 50 The specific results can be seen Figure 2 , Figure 3 and Figure 4 ,in Figure 2 It is the IC of goat milk casein enzymatic hydrolysate No. 1 50 Value result graph, Figure 3 It is the IC of goat milk casein enzymatic hydrolysate No. 2 50 Value result graph, Figure 4 It is the IC of goat milk casein enzymatic hydrolysate No. 3 50 Value result graph.

[0082] Depend on Figure 2 , Figure 3 and Figure 4 It can be seen that the ability of goat milk casein enzymatic hydrolysates 1-3 to inhibit superoxide anion is concentration-dependent. The IC values ​​of goat milk casein enzymatic hydrolysates 1-3 were obtained by calculation. 50 The values ​​were 36.64 mg / mL, 44.27 mg / mL and 56.15 mg / mL, respectively. Subsequently, the goat casein hydrolysates 1-4 were detected and analyzed by liquid chromatography-mass spectrometry, and the mass contents of 8 candidate peptides in goat casein hydrolysates 1-4 were obtained. The specific contents can be seen in Table 5. It can be found that the relative contents of peptide PWP, peptide MPLW, peptide PLW and peptide LPW in goat casein hydrolysates 1-4 are 0.08%, 0.65%, 0.09% and 0.37% and above, respectively. The relative contents are high and stable, which plays an important role in the antioxidant activity of goat casein hydrolysates.

[0083] Table 4

[0084]

[0085] Table 5

[0086]

[0087] 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 goat milk casein enzymatic hydrolysate, characterized in that: The goat milk casein enzymatic hydrolysate includes peptide segment PWP, peptide segment MPLW, peptide segment PLW, and peptide segment LPW; Based on the mass of the goat casein enzymatic hydrolysate, the content of the peptide segment PWP is ≥0.08%, the content of the peptide segment MPLW is ≥0.72%, the content of the peptide segment PLW is ≥0.13%, and the content of the peptide segment LPW is ≥0.94%.

2. The goat milk casein enzymatic hydrolysate according to claim 1, characterized in that The degree of hydrolysis of the goat milk casein enzymatic hydrolysate is 10%-15%; and / or, The mass content of peptides with a molecular weight of ≤3000 Da in the goat milk casein enzymatic hydrolysate is greater than 90%, and the mass content of peptides with a molecular weight of <1000 Da is greater than 60%.

3. The goat milk casein enzymatic hydrolysate according to claim 1, characterized in that The goat milk casein enzymatic hydrolysate is obtained by enzymatically hydrolyzing goat milk casein as a raw material; The enzymatic treatment comprises using trypsin; or, The enzymatic treatment comprises using trypsin and alkaline protease; or, The enzymatic treatment includes the use of trypsin, bromelain and flavor protease.

4. The method for preparing the goat casein enzymatic hydrolysate according to any one of claims 1 to 3, characterized in that: include: Using protease to enzymatically hydrolyze goat milk casein to obtain the goat milk casein enzymatic hydrolysate; The protease comprises trypsin; or, The proteases include trypsin and alkaline protease; or, The proteases include trypsin, bromelain and flavorin.

5. The preparation method according to claim 4, characterized in that: Based on each gram of goat milk casein, the enzyme activity of the trypsin is 4000 U, and the addition amount is 0.05-0.6wt%; the enzyme activity of the alkaline protease is 200000 U, and the addition amount is 0.1-0.6wt%; the enzyme activity of the bromelain is 50000 U, and the addition amount is 0.1-0.6wt%; the enzyme activity of the flavor protease is 20000 U, and the addition amount is 0.1-0.6wt%.

6. The preparation method according to claim 4, characterized in that: The enzymatic hydrolysis temperature is 40-50° C., the time is 1.5-5.0 h, and the pH value is 7.0-9.

0.

7. Use of the goat milk casein enzymatic hydrolysate according to any one of claims 1 to 3 or the goat milk casein enzymatic hydrolysate prepared by the preparation method according to any one of claims 4 to 6 in the preparation of antioxidant health products.

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