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 a lipid-lowering effect, which is mainly achieved by regulating the types and contents of functional peptides therein. Among them, the functional peptides include peptide segments FDSWPCVMGR, peptide segment LPW, peptide segment FAWPQYLK and peptide segment KLF, which have biological activity, the potential to bind to 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), and the efficacy of inhibiting pancreatic lipase, thereby having a lipid-lowering 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] Currently, drugs widely used to treat hyperlipidemia, such as statins, are drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase to reduce low-density lipoprotein cholesterol, thereby preventing and treating hyperlipidemia and cardiovascular diseases. Although they are very effective in preventing and treating hyperlipidemia, they also bring some side effects that affect human health, such as liver damage, muscle damage, kidney damage, and effects on patients' blood sugar levels. Therefore, how to screen and obtain safe and efficient substances with lipid-lowering effects is an urgent problem to be solved. Summary of the invention

[0003] The invention provides a goat casein enzymatic hydrolysate, which has low cost and has the effect of lowering blood lipid.

[0004] 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.

[0005] The present invention also 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 blood lipid-lowering related products.

[0006] The present invention provides a goat milk casein enzymatic hydrolysate, wherein the goat milk casein enzymatic hydrolysate comprises a peptide segment FDSWPCVMGR, a peptide segment LPW, a peptide segment FAWPQYLK and a peptide segment KLF;

[0007] Based on the mass of goat casein enzymatic hydrolysate, the content of peptide FDSWPCVMGR is ≥0.01%, the content of peptide LPW is ≥0.37%, the content of peptide FAWPQYLK is ≥0.91%, and the content of peptide KLF is ≥0.01%.

[0008] 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,

[0009] 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%.

[0010] 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;

[0011] The enzymatic treatment comprises using at least one of trypsin, alkaline protease, bromelain and flavor protease.

[0012] 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;

[0013] The protease includes at least one of trypsin, alkaline protease, bromelain and flavor protease.

[0014] 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%.

[0015] 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.

[0016] The preparation method as described above, wherein the protease comprises trypsin.

[0017] The present invention also 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 blood lipid-lowering related products.

[0018] The present invention provides a goat casein enzymatic hydrolysate, which has a lipid-lowering effect, which is mainly achieved by regulating the types and contents of functional peptides therein. Among them, the functional peptides include peptide segments FDSWPCVMGR, peptide segment LPW, peptide segment FAWPQYLK and peptide segment KLF, which have biological activity, the potential to bind to 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), and the efficacy of inhibiting pancreatic lipase. HMG-CoA reductase is the rate-limiting enzyme in the process of cholesterol synthesis in liver cells in animals and humans, so binding to HMG-CoA reductase can inhibit the enzyme from functioning and thus hinder cholesterol synthesis. Pancreatic lipase is responsible for enzymatically hydrolyzing triglycerides in the diet into diglycerols, monoglycerols, glycerol and fatty acids to promote fat absorption, so inhibiting the activity of pancreatic lipase can reduce the probability of triglycerides in the diet being enzymatically hydrolyzed and absorbed, thereby playing a lipid-lowering effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram showing the degree of hydrolysis of the goat milk casein enzymatic hydrolysate in Example 2;

[0020] Figure 2 is the IC of goat milk casein enzymatic hydrolysate No. 1 in Example 3 50 Value result graph;

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

[0022] Figure 4 is the IC of goat milk casein enzymatic hydrolysate No. 3 in Example 3 50 Value result graph. DETAILED DESCRIPTION

[0023] 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.

[0024] 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 FDSWPCVMGR, a peptide segment LPW, a peptide segment FAWPQYLK and a peptide segment KLF;

[0025] Based on the mass of goat casein enzymatic hydrolysate, the content of peptide FDSWPCVMGR is ≥0.01%, the content of peptide LPW is ≥0.37%, the content of peptide FAWPQYLK is ≥0.91%, and the content of peptide KLF is ≥0.01%.

[0026] 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 also provides a low-cost goat milk casein hydrolysate, which includes peptide segments FDSWPCVMGR, peptide segment LPW, peptide segment FAWPQYLK and peptide segment KLF, which has the effect of lowering blood lipids.

[0027] It can be understood that the peptide segment FDSWPCVMGR is a decapeptide segment (Phe-Asp-Ser-Trp-Pro-Cys-Val-Met-Gly-Arg) consisting of phenylalanine, aspartic acid, serine, tryptophan, proline, cysteine, valine, methionine, glycine, and arginine from N-terminus to C-terminus, the peptide segment LPW is a tripeptide segment (Leu-Pro-Trp) consisting of leucine, proline, and tryptophan from N-terminus to C-terminus, the peptide segment FAWPQYLK is an octapeptide segment (Phe-Ala-Trp-Pro-Gln-Tyr-Leu-Lys) consisting of phenylalanine, alanine, tryptophan, proline, glutamine, tyrosine, leucine, and lysine from N-terminus to C-terminus, and the peptide segment KLF is a tripeptide segment (Lys-Leu-Phe) consisting of lysine, leucine, and phenylalanine from N-terminus to C-terminus.

[0028] In addition, in the present invention, based on the quality of goat casein enzymatic hydrolysate, the contents of peptides FDSWPCVMGR, peptide LPW, peptide FAWPQYLK and peptide KLF are limited to meet the above ranges, which can enable the goat casein enzymatic hydrolysate to show good efficacy in lowering blood lipids.

[0029] After long-term research, it was found that the goat casein enzymatic hydrolysate of the present invention has a lipid-lowering effect, which is mainly achieved by regulating the types and contents of functional peptides therein. Peptide segments FDSWPCVMGR, peptide segment LPW, peptide segment FAWPQYLK and peptide segment KLF have biological activity and have the potential to bind to 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). HMG-CoA reductase is the rate-limiting enzyme in the process of cholesterol synthesis in hepatocytes in animals and humans. Therefore, binding to HMG-CoA reductase can inhibit the enzyme from functioning and thus hinder cholesterol synthesis. At the same time, peptide segments FDSWPCVMGR, peptide segment LPW, peptide segment FAWPQYLK and peptide segment KLF also have a good inhibitory effect on pancreatic lipase. Since pancreatic lipase is responsible for enzymatically hydrolyzing triglycerides in the diet into diglycerols, monoglycerols, glycerol and fatty acids to promote fat absorption, inhibiting the activity of pancreatic lipase can reduce the probability of triglycerides in the diet being hydrolyzed and absorbed, thereby playing a lipid-lowering role.

[0030] 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.

[0031] 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.

[0032] 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 prepared 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 hypolipidemic effect of goat milk casein hydrolysate.

[0033] 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.

[0034] A lot of research has been conducted on how to make the enzymatic hydrolyzate of goat milk casein contain the expected functional peptides, that is, the peptides FDSWPCVMGR, LPW, FAWPQYLK and KLF, and the content of which meets the above requirements, and 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 the peptides FDSWPCVMGR, LPW, FAWPQYLK and KLF, and the peptide content meets the above requirements. By limiting the preparation method of the protease, the goat milk cheese hydrolyzate containing the expected functional peptides can be obtained simply and quickly, which is convenient for expanding production.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In aspects of the present invention, the protease comprises trypsin.

[0043] After a large number of experiments, it was found that when goat casein is hydrolyzed with protease to obtain goat casein enzymatic hydrolysate, if the protease includes trypsin, the lipid-lowering effect of the goat casein enzymatic hydrolysate can be significantly improved.

[0044] 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 obtained goat casein enzymatic hydrolysate has an inhibition rate of >40% in the pancreatic lipase inhibition experiment; and the protease is at least one of alkaline protease, bromelain, and flavor protease, and the obtained goat casein enzymatic hydrolysate has an inhibition rate of 5%-20% in the pancreatic lipase inhibition experiment.

[0045] 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.

[0046] 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 lipid-lowering related products.

[0047] It is understandable that blood lipid-lowering related products include but are not limited to food, health products, medicines and blood lipid-lowering detection products. A large amount of research data proves that the goat casein enzymatic hydrolysate provided by the present invention, including peptides FDSWPCVMGR, peptides LPW, peptides FAWPQYLK and peptides KLF, and the peptide content meets the aforementioned requirements, has an obvious blood lipid-lowering effect, and can not only be applied to food and health products in the conventional sense, such as blood lipid-lowering foods or blood lipid-lowering health products, but also can be used to prepare blood lipid-lowering drugs, etc., thereby broadening the scope of application of goat casein enzymatic hydrolysate, and also providing new raw materials for blood lipid-lowering products.

[0048] The technical solution of the present application will be further explained below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The reagents used are commercially available or available from public channels unless otherwise specified.

[0049] Example 1: Preparation of goat milk casein enzyme hydrolysate

[0050] (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.

[0051] (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.

[0052] (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.

[0053] (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.

[0054] Example 2: Detection of goat milk casein hydrolysate

[0055] (1) Hydrolysis degree detection of goat milk casein hydrolysate

[0056] 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:

[0057]

[0058] Where h is the number of peptide bonds hydrolyzed, mmol / g; h totis 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.

[0059] The specific degree of hydrolysis can be seen Figure 1 Among them, samples 1-4 correspond to goat casein enzymatic hydrolysates No. 1-4 respectively, the hydrolysis degree of goat casein enzymatic hydrolysate No. 1 is 10.13%, the hydrolysis degree of goat casein enzymatic hydrolysate No. 2 is 11.15%, the hydrolysis degree of goat casein enzymatic hydrolysate No. 3 is 12.33%, and the hydrolysis degree of goat casein enzymatic hydrolysate No. 4 is 12.22%.

[0060] (2) Molecular weight distribution detection of goat milk casein hydrolysate

[0061] 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%.

[0062] Table 1

[0063]

[0064] Example 3: Screening of lipid-lowering functional peptides

[0065] (1) LC-MS / MS mass spectrometry identification

[0066] 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.

[0067] 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.

[0068] 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.

[0069] (2) Bioactivity screening

[0070] 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.

[0071] Table 2

[0072]

[0073] (3) Molecular docking screening

[0074] In order to screen functional peptides with lipid-lowering effects, the above bioactive peptides were molecularly docked with 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) (PDBID: 1HWK). According to the binding energy score, the number of hydrogen bonds shown in the wiring diagram, and the degree of reorganization with the HMG-CoA reductase binding site, 70 bioactive peptides were screened, and the binding energy scores of the bioactive peptides can be seen in Table 3. HMG-CoA reductase is the rate-limiting enzyme in the process of cholesterol synthesis in hepatocytes in animals and humans. Inhibiting HMG-CoA reductase can hinder cholesterol synthesis.

[0075] Through the binding energy score and the content distribution of bioactive peptides in goat casein enzymatic hydrolysate, 10 candidate functional peptides were screened, namely peptide 14 (FDSWPCVMGR), peptide 25 (LPW), peptide 29 (GPFPIL), peptide 30 (LPFL), peptide 35 (LFL), peptide 37 (FLY), peptide 46 (LAF), peptide 47 (CR), peptide 56 (FAWPQYLK), and peptide 69 (KLF).

[0076] Table 3

[0077]

[0078]

[0079] (4) Functional verification of candidate functional peptides

[0080] Since pancreatic lipase is responsible for enzymatically hydrolyzing triglycerides in the diet into diglycerols, monoglycerols, glycerol and fatty acids to promote fat absorption, inhibiting the activity of pancreatic lipase can effectively reduce the probability of triglycerides in the diet being hydrolyzed and absorbed, thereby playing a role in lowering blood lipids. The present invention screens functional peptides with a lipid-lowering effect by detecting the activity of candidate functional peptides in inhibiting pancreatic lipase in vitro.

[0081] The above 10 candidate functional peptides were synthesized by Fmoc solid phase organic synthesis method, and the purity of the candidate functional peptides was >97%. The sheep milk casein hydrolysate No. 1-4 obtained in Example 1, the 10 candidate functional peptides synthesized, and the lipid-lowering drug orlistat as a positive control were dissolved and diluted to a final concentration of 1 mg / mL using 0.1 mol / L Tris-HCl buffer with a pH value of 8.2 to obtain a sample to be tested. Pancreatic lipase was dissolved and diluted to 5 mg / mL using distilled water to obtain a pancreatic lipase solution. Para-nitrobenzene laurate was dissolved using a 0.05 mol / L sodium acetate aqueous solution containing 1% Triton X-100 to obtain a 0.1% (w / v) para-nitrobenzene laurate solution, which was placed in hot water to accelerate dissolution, and cooled to room temperature for use after complete dissolution. Add 250 μL of the above 0.1% (w / v) p-nitrobenzene laurate solution, 100 μL of the sample to be tested, and 200 μL of 0.1 mol / L Tris-HCl buffer with a pH value of 8.2 to the centrifuge tube, and finally add 150 μL of pancreatic lipase solution for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 37°C and the enzymatic hydrolysis time is 2 h to obtain the enzymatic hydrolyzate. The enzymatic hydrolyzate is centrifuged at 10000 r / min for 1 min, and the absorbance value of the supernatant is measured at 420 nm. Among them, the blank control operation is similar to the sample group to be tested, the only difference is that the sample to be tested is replaced by 0.1 mol / L Tris-HCl buffer with a pH value of 8.2; the sample control operation is similar to the sample group to be tested, the only difference is that 100 μL of the sample to be tested is deleted. The inhibition rate of pancreatic lipase can be calculated based on the measured absorbance value, and the calculation formula is as follows: (1-(A1-A2) / A0)×100%, where A1 is the absorbance value of the supernatant of the sample to be tested, A2 is the absorbance value of the supernatant of the sample control, and A0 is the absorbance value of the supernatant of the blank control. The specific results are shown in Table 4.

[0082] As shown in Table 4, among the candidate functional peptides, the inhibition rate of peptide 25 (LPW) can reach 69.91% ± 5.89%, and the inhibition rate of peptide 56 (FAWPQYLK) can reach 71.89% ± 0.41%, which has a good inhibitory effect; peptide 14 (FDSWPCVMGR) and peptide 69 (KLF) also have a certain inhibitory effect, and their inhibition rates are 16.11% ± 1.74% and 11.57% ± 0.85%, respectively. In the goat milk casein hydrolysate, the inhibition rate of goat milk casein hydrolysate No. 1-3 is close to 50%, and the inhibition rate of goat milk casein hydrolysate No. 4 is slightly weaker, reaching 12.56% ± 2.85%, indicating that the use of trypsin for enzymatic hydrolysis in the preparation of goat milk casein hydrolysate has a more significant promoting effect on the inhibitory activity of pancreatic lipase.

[0083] According to the inhibition rate results, goat milk casein enzymatic hydrolysates 1-3 were selected for concentration IC 50 Detection. Use 0.1 mol / L Tris-HCl buffer with a pH value of 8.2 to dissolve and dilute goat casein enzymatic hydrolysates 1-3 to a final concentration of 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL and 4 mg / mL, respectively, to obtain a series of samples to be tested. Add 250 μL of the above-mentioned 0.1% (w / v) p-nitrobenzene laurate solution, 100 μL of the sample to be tested and 200 μL of 0.1 mol / L Tris-HCl buffer with a pH value of 8.2 into a centrifuge tube, and finally add 150 μL of pancreatic lipase solution for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 37°C and the enzymatic hydrolysis time is 2 h to obtain an enzymatic hydrolyzate. The enzymatic hydrolyzate was centrifuged at 10,000 r / min for 1 min, and the absorbance value of the supernatant was measured at 420 nm. 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.

[0084] Depend on Figure 2 , Figure 3 and Figure 4 It can be seen that the pancreatic lipase inhibitory activity of goat milk casein enzymatic hydrolysates No. 1-3 showed a concentration-dependent relationship. The IC values ​​of goat milk casein enzymatic hydrolysates No. 1-3 were obtained by calculation. 50 The values ​​were 0.87 mg / mL, 1.42 mg / mL and 1.25 mg / mL, respectively. Subsequently, the goat casein hydrolysates 1-4 were detected and analyzed by liquid chromatography-mass spectrometry, and the mass contents of 20 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 FDSWPCVMGR, peptide LPW, peptide FAWPQYLK and peptide KLF in goat casein hydrolysates 1-4 are 0.01%, 0.37%, 0.91%, 0.01% and above, respectively. The relative contents are high and stable, which plays an important role in the lipid-lowering activity of goat casein hydrolysates.

[0085] Table 4

[0086]

[0087] Table 5

[0088]

[0089] 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 comprises peptide segment FDSWPCVMGR, peptide segment LPW, peptide segment FAWPQYLK and peptide segment KLF; Based on the mass of the goat casein enzymatic hydrolysate, the content of the peptide segment FDSWPCVMGR is ≥0.01%, the content of the peptide segment LPW is ≥0.37%, the content of the peptide segment FAWPQYLK is ≥0.91%, and the content of the peptide segment KLF is ≥0.01%.

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 comprises using trypsin, bromelain and flavor protease; or, The enzymatic treatment includes using alkaline protease and flavor protease.

4. A 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 flavor protease; or, The proteases include alkaline proteases and flavor proteases.

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. The preparation method according to claim 4, characterized in that: The protease includes trypsin.

8. Use of the goat casein enzymatic hydrolysate according to any one of claims 1 to 3 or the goat casein enzymatic hydrolysate prepared by the preparation method according to any one of claims 4 to 7 in the preparation of products related to lowering blood lipids.

Images