Functional peptide with hypoglycemic effect, preparation method and application thereof

Abstract

The present invention provides a functional peptide with hypoglycemic effect, a preparation method and application. The functional peptide provided by the present invention includes the peptide segment FYPQLFR, which has biological activity, has the potential to bind to dipeptidyl peptidase 4 (DPP-4), and can also inhibit α-amylase. Among them, DPP-4 is a serine protease on the cell surface, which can hydrolyze glucagon-like peptide-1 (GLP-1), thereby inhibiting the secretion of insulin by pancreatic β cells and promoting the secretion of glucagon by pancreatic α cells, causing blood sugar levels to rise. Therefore, the functional peptide of the present invention can play a hypoglycemic effect by binding to and inhibiting DPP-4. α-Amylase is a key enzyme in the starch hydrolysis process, which can catalyze the hydrolysis of α-1,4-glycosidic bonds in starch. Its activity is closely related to the increase in postprandial blood sugar levels. Therefore, the functional peptide of the present invention can also inhibit the activity of α-amylase to regulate postprandial blood sugar fluctuations.

Description

Technical Field

[0001] The invention relates to biotechnology, and in particular to a functional peptide with blood sugar lowering effect, a preparation method and application thereof. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar, which may cause chronic damage and dysfunction of various organs and tissues in the body, including the kidneys, heart, blood vessels, eyes, nerves and extremities. Diabetes has become an important challenge in my country's public health field, and effective prevention and control measures are needed to deal with this increasingly serious health problem.

[0003] Controlling the occurrence and development of diabetes mainly depends on controlling blood sugar. Currently, the drugs used to lower or control blood sugar mainly include sulfonamides, dimethoate, benzoic acid derivatives, glycosidase inhibitors and thiazolidinediones. Common hypoglycemic drugs such as metformin and sulfonylureas have side effects to varying degrees and are extremely unfriendly to the bodies of patients with hyperglycemia and diabetes. For example, metformin, as a first-line treatment drug, may cause gastrointestinal reactions (such as nausea, vomiting, abdominal distension, diarrhea), lactic acidosis (especially in patients with renal insufficiency), and affected vitamin B12 absorption. Sulfonylureas may cause hypoglycemia, weight gain, skin allergic reactions, leukopenia, and may cause secondary failure. Therefore, how to safely and effectively lower blood sugar has become the focus of attention in my country and even the world. Summary of the invention

[0004] The present invention provides a functional peptide with a blood sugar lowering effect. The functional peptide has biological activity, has the potential to bind to dipeptidyl peptidase 4 (DPP-4), and also has a good inhibitory effect on α-amylase, thereby having the effect of lowering blood sugar.

[0005] The invention provides a goat casein enzymatic hydrolysate, comprising the above functional peptides, which has low cost and has the effect of lowering blood sugar.

[0006] 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.

[0007] The present invention also provides a use of the functional peptide, the goat casein enzymatic hydrolysate or the goat casein enzymatic hydrolysate prepared by the preparation method in the preparation of blood sugar-lowering related products.

[0008] The present invention provides a functional peptide with blood sugar lowering effect, wherein the functional peptide comprises the peptide segment FYPQLFR.

[0009] The functional peptide as described above, wherein the functional peptide further includes at least one of the peptide segment FFF, the peptide segment FFR, the peptide segment FFY, the peptide segment MPLW, the peptide segment LPW, the peptide segment FLY, the peptide segment QFY, and the peptide segment AWPQYLK.

[0010] The present invention provides a goat casein enzymatic hydrolysate, wherein the goat casein enzymatic hydrolysate comprises the above-mentioned functional peptides.

[0011] The goat casein enzymatic hydrolysate as described above, wherein, based on the mass of the goat casein enzymatic hydrolysate, the content of the peptide FYPQLFR is ≥0.08%, the content of the peptide FFF is ≥0.01%, the content of the peptide FFR is ≥0.01%, the content of the peptide FFY is ≥0.01%, the content of the peptide MPLW is ≥0.92%, the content of the peptide LPW is ≥0.37%, the content of the peptide FLY is ≥0.03%, the content of the peptide QFY is ≥1.37%, and the content of the peptide AWPQYLK is ≥0.03%.

[0012] 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,

[0013] 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%.

[0014] 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;

[0015] The enzymatic treatment comprises using at least one of trypsin, alkaline protease, bromelain and flavor protease.

[0016] 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;

[0017] The protease includes at least one of trypsin, alkaline protease, bromelain and flavor protease.

[0018] 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%.

[0019] 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.

[0020] The preparation method as described above, wherein the protease comprises trypsin.

[0021] The present invention also provides a use of the functional peptide, the goat casein enzymatic hydrolysate or the goat casein enzymatic hydrolysate prepared by the preparation method in the preparation of blood sugar-lowering related products.

[0022] The present invention provides a functional peptide with hypoglycemic effect, which has biological activity and has the potential to bind to dipeptidyl peptidase 4 (DPP-4). DPP-4 is a serine protease on the cell surface, which can hydrolyze glucagon-like peptide-1 (GLP-1), thereby inhibiting the secretion of insulin by pancreatic β cells and promoting the secretion of glucagon by pancreatic α cells, resulting in an increase in blood sugar levels. Therefore, the functional peptide of the present invention is combined with DPP-4, indicating that it can inhibit the function of DPP-4, thereby playing a role in lowering blood sugar. At the same time, the functional peptide of the present invention also has a good inhibitory effect on α-amylase, which is a key enzyme in the starch hydrolysis process and can catalyze the hydrolysis of α-1,4-glycosidic bonds in starch. Its activity is closely related to the increase in postprandial blood sugar levels. Therefore, inhibiting the activity of α-amylase can effectively regulate postprandial blood sugar fluctuations, which is of great significance for lowering blood sugar. In addition, the functional peptide of the present invention also has the advantages of definite ingredients, high safety, non-allergenicity, high activity and easy absorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram showing the degree of hydrolysis of the goat milk casein enzymatic hydrolysate in Example 2;

[0024] Figure 2 This is the molecular docking diagram of the peptide FYPQLFR (red) and DPP-4 (blue) in Example 3;

[0025] Figure 3 is the IC of peptide FYPQLFR in Example 3 50 Value result graph;

[0026] Figure 4 is the IC of goat milk casein enzymatic hydrolysate No. 1 in Example 3 50 Value result graph;

[0027] Figure 5 is the IC of goat milk casein enzymatic hydrolysate No. 2 in Example 3 50 Value result graph;

[0028] Figure 6 is the IC of goat milk casein enzymatic hydrolysate No. 3 in Example 3 50Value result graph. DETAILED DESCRIPTION

[0029] 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.

[0030] The first aspect of the present invention provides a functional peptide with hypoglycemic effect, wherein the functional peptide includes a peptide segment FYPQLFR. The peptide segment FYPQLFR is a heptapeptide segment (Phe-Tyr-Pro-Gln-Leu-Phe-Arg) consisting of phenylalanine, tyrosine, proline, glutamine, leucine, phenylalanine, and arginine from the N-terminus to the C-terminus.

[0031] The functional peptide provided by the present invention has biological activity and has the potential to bind to dipeptidyl peptidase 4 (DPP-4). DPP-4 is a serine protease on the cell surface that can hydrolyze glucagon-like peptide-1 (GLP-1), thereby inhibiting the secretion of insulin by pancreatic β cells and promoting the secretion of glucagon by pancreatic α cells, resulting in an increase in blood sugar levels. Therefore, the functional peptide of the present invention binds to DPP-4, indicating that it can inhibit the function of DPP-4, thereby playing a role in lowering blood sugar. At the same time, the functional peptide of the present invention also has a good inhibitory effect on α-amylase, which is a key enzyme in the starch hydrolysis process and can catalyze the hydrolysis of α-1,4-glycosidic bonds in starch. Its activity is closely related to the increase in postprandial blood sugar levels. Therefore, inhibiting the activity of α-amylase can effectively regulate postprandial blood sugar fluctuations, which is of great significance for lowering blood sugar. In addition, as a polypeptide, the functional peptide of the present invention also has the advantages of a certain composition of the polypeptide itself, high safety, low allergy, high activity, and easy absorption.

[0032] In a specific embodiment, the functional peptide further includes at least one of the peptide segment FFF, the peptide segment FFR, the peptide segment FFY, the peptide segment MPLW, the peptide segment LPW, the peptide segment FLY, the peptide segment QFY, and the peptide segment AWPQYLK.

[0033] The peptide FFF is a tripeptide consisting of phenylalanine, phenylalanine, and phenylalanine from the N-terminus to the C-terminus (Phe-Phe-Phe), the peptide FFR is a tripeptide consisting of phenylalanine, phenylalanine, and arginine from the N-terminus to the C-terminus (Phe-Phe-Arg), the peptide FFY is a tripeptide consisting of phenylalanine, phenylalanine, and tyrosine from the N-terminus to the C-terminus (Phe-Phe-Tyr), the peptide MPLW is a tetrapeptide consisting of methionine, proline, leucine, and tryptophan from the N-terminus to the C-terminus (Met-Pro-Leu-Trp), and the peptide LPW is a tripeptide consisting of phenylalanine, phenylalanine, and arginine from the N-terminus to the C-terminus (Phe-Phe-Arg). The peptide segment AWPQYLK is a heptapeptide segment (Ala-Trp-Pro-Gln-Tyr-Leu-Lys) consisting of alanine, tryptophan, proline, glutamine, tyrosine, leucine, and lysine from N-terminus to C-terminus.

[0034] The above functional peptides also have biological activity, not only can bind to DPP-4 and inhibit it, but also can inhibit the activity of α-amylase, thereby playing a role in lowering blood sugar. When used together with FYPQLFR, it can exert a better blood sugar lowering effect. The second aspect of the present invention provides a goat milk casein hydrolysate, including the above functional peptides. Goat milk casein hydrolysates (GMCH) refer to the enzymatic hydrolysate obtained by enzymatic hydrolysis of goat milk casein. Although functional peptides have the advantages of determined ingredients, high safety, non-allergenicity, high activity and easy absorption when used alone, the cost of separation and purification 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 the above functional peptides and thus also has the effect of lowering blood sugar.

[0035] Further, based on the quality of goat casein enzymatic hydrolysate, the content of peptide FYPQLFR is ≥0.08%, the content of peptide FFF is ≥0.01%, the content of peptide FFR is ≥0.01%, the content of peptide FFY is ≥0.01%, the content of peptide MPLW is ≥0.92%, the content of peptide LPW is ≥0.37%, the content of peptide FLY is ≥0.03%, the content of peptide QFY is ≥1.37%, and the content of peptide AWPQYLK is ≥0.03%. The present invention can further show good efficacy in lowering blood sugar by regulating the content of functional peptides in goat casein enzymatic hydrolysate.

[0036] 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.

[0037] 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.

[0038] 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 casein, while limiting the type of enzyme preparation to meet the above range, functional peptides can be further enriched to improve the hypoglycemic effect of goat casein enzymolysis products.

[0039] 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.

[0040] A lot of research has been done on how to make the enzymatic hydrolyzate of goat milk casein contain the expected functional peptides, and it has been proved that the selection of protease has a key influence on the result. Enzymatic hydrolysis of goat milk casein with at least one of trypsin, alkaline protease, bromelain, and flavor protease can obtain a goat milk casein hydrolyzate with the expected functional peptides. 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] In aspects of the present invention, the protease comprises trypsin.

[0049] 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 hypoglycemic effect of the goat casein enzymatic hydrolysate can be significantly improved.

[0050] 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 >50% in the α-amylase 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 15%-25% in the α-amylase inhibition experiment.

[0051] 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.

[0052] The fourth aspect of the present invention provides a use of the above functional peptide, the above goat casein enzymatic hydrolysate or the goat casein enzymatic hydrolysate prepared by the above preparation method in the preparation of hypoglycemic drugs.

[0053] A large amount of research data proves that the functional peptides provided by the present invention and the goat casein enzymatic hydrolysate including the functional peptides have obvious hypoglycemic effects and can be used to prepare hypoglycemic drugs, etc., thereby broadening the application scope of the functional peptides and providing new raw materials for hypoglycemic drugs.

[0054] Furthermore, according to the amino acid sequence of the functional peptide provided by the present invention, those skilled in the art can obtain the polynucleotide sequence encoding the amino acid sequence according to conventional techniques in the art, and can also obtain the expression vector containing the polynucleotide sequence, and then obtain the recombinant cell containing the polynucleotide sequence and / or the expression vector.

[0055] It is understandable that, based on the known amino acid sequence of the functional peptide of the present invention, a person skilled in the art can obtain a polynucleotide encoding the amino acid sequence of the functional peptide, insert the polynucleotide into an expression vector to obtain a recombinant expression vector, and then transform the recombinant expression vector into a host cell to obtain a recombinant cell.

[0056] Therefore, the present invention provides a polynucleotide encoding the amino acid sequence of the functional peptide mentioned above; the present invention provides a recombinant expression vector comprising the polynucleotide mentioned above; the present invention also provides a recombinant cell comprising the recombinant expression vector mentioned above.

[0057] 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.

[0058] Example 1: Preparation of goat milk casein enzyme hydrolysate

[0059] (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.

[0060] (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.

[0061] (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.

[0062] (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.

[0063] Example 2: Detection of goat milk casein hydrolysate

[0064] (1) Hydrolysis degree detection of goat milk casein hydrolysate

[0065] 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:

[0066]

[0067] 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.

[0068] 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%.

[0069] (2) Molecular weight distribution detection of goat milk casein hydrolysate

[0070] 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%.

[0071] Table 1

[0072]

[0073] Example 3: Screening of hypoglycemic functional peptides

[0074] (1) LC-MS / MS mass spectrometry identification

[0075] 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.

[0076] 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.

[0077] 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.

[0078] (2) Bioactivity screening

[0079] 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.

[0080] Table 2

[0081]

[0082] (3) Molecular docking screening

[0083] In order to screen functional peptides with hypoglycemic effect, the above bioactive peptides were molecularly docked with dipeptidyl peptidase 4 (DPP-4) (PDBID: 5I7U). According to the binding energy score, the number of hydrogen bonds shown in the wiring diagram, and the degree of reorganization with the DPP-4 binding site, 63 bioactive peptides were screened. The binding energy scores of the bioactive peptides can be seen in Tables 3 and Figure 2 , Figure 2 This is the molecular docking diagram of peptide 31 (FYPQLFR) (red) and DPP-4 (blue). The green part in the figure is the secondary structure of the protein. The yellow dotted line and the numbers next to the dotted line represent the hydrogen bonds and distances formed between atoms (in angstroms). The capital letters and numbers in the figure are the abbreviations and positions of amino acids. DPP-4 is a serine protease on the cell surface that can hydrolyze glucagon-like peptide-1 (GLP-1), thereby inhibiting the secretion of insulin by pancreatic β cells and promoting the secretion of glucagon by pancreatic α cells, resulting in increased blood sugar levels. Therefore, if the bioactive peptide can bind to DPP-4, it means that it can inhibit the function of DPP-4, thereby playing a role in lowering blood sugar.

[0084] Through the binding energy score and the content distribution of bioactive peptides in goat casein enzymatic hydrolysate, 9 candidate functional peptides were screened, namely peptide 2 (FFF), peptide 4 (FFR), peptide 6 (FFY), peptide 15 (MPLW), peptide 25 (LPW), peptide 31 (FYPQLFR), peptide 37 (FLY), peptide 44 (QFY), and peptide 62 (AWPQYLK).

[0085] Table 3

[0086]

[0087] (4) Functional verification of candidate functional peptides

[0088] α-amylase is a key enzyme in the starch hydrolysis process, which can catalyze the hydrolysis of α-1,4-glycosidic bonds in starch. Its activity is closely related to the increase in blood sugar levels after a meal. Therefore, inhibiting the activity of α-amylase can effectively regulate postprandial blood sugar fluctuations, which is of great significance for lowering blood sugar. The present invention screens functional peptides with hypoglycemic effects by detecting the activity of candidate functional peptides in inhibiting α-amylase in vitro.

[0089] The above 9 candidate functional peptides were synthesized by Fmoc solid phase organic synthesis method, and the purity of the candidate functional peptides was >97%. The goat milk casein hydrolysate No. 1-4 obtained in Example 1 and the 9 candidate functional peptides synthesized were dissolved and diluted to a final concentration of 1 mg / mL using 0.1 mol / L phosphate buffer with a pH value of 6.9 to obtain samples to be tested. 45 μL of the sample to be tested and 15 μL of 0.02 mg / mL α-amylase solution were added to a 96-well plate, placed in a 37°C water bath for reaction for 10 min, and then 60 μL of 0.08wt% starch solution was added, placed in a 37°C water bath for reaction for 15 min, and then 60 μL of 1.0 mol / L hydrochloric acid was added to terminate the reaction, and then 20 μL of 0.01 mol / L iodine solution was added for 5 min of color development reaction, and after the reaction, 60 μL of 1 mol / L hydrochloric acid was added to terminate the reaction, and then the absorbance value was measured on an ELISA instrument at a wavelength of 630 nm. Among them, a sample blank group is set to correct the nonspecific absorbance of the sample group to be tested. The operation can refer to the sample group to be tested, and the only difference is that the α-amylase solution is deleted; a positive control group is set to determine the maximum activity of α-amylase in the absence of the sample to be tested. The operation can refer to the sample group to be tested, and the only difference is that the sample to be tested is replaced by metformin; a blank group is set to correct the background absorbance. The operation can refer to the sample group to be tested, and the only difference is that the sample to be tested, α-amylase solution and starch solution are deleted, and only phosphate buffer and iodine solution are retained. The inhibition rate of α-amylase can be calculated based on the measured absorbance value. The calculation formula is as follows: (1-(A sb -A sc ) / (A b- A c ))×100%, where A sb is the absorbance value of the sample group to be tested, A sc is the absorbance value of the sample blank group, A b is the absorbance value of the positive control group, A c is the absorbance value of the blank group. The specific results are shown in Table 4.

[0090] As shown in Table 4, among the candidate functional peptides, peptide 31 (FYPQLFR) has a strong inhibitory effect, and its inhibition rate can reach 30.69%±0.53%. Among the goat milk casein enzymatic hydrolysates, the inhibition rates of goat milk casein enzymatic hydrolysates 1-3 are all over 50%, and the inhibition rate of goat milk casein enzymatic hydrolysate 4 is slightly weaker, reaching 19.03%±2.46%, indicating that the use of trypsin for enzymatic hydrolysis in the preparation of goat milk casein enzymatic hydrolysates has a more significant promoting effect on the α-amylase inhibitory activity.

[0091] According to the inhibition rate results, the concentration IC of peptide 31 (FYPQLFR) was selected for in vitro inhibition of α-amylase 50 The final concentrations of peptide 31 (FYPQLFR) were set to 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL and 4 mg / mL to obtain a series of samples to be tested, and then the α-amylase inhibition experiment was performed. The specific results can be seen in Figure 3 .

[0092] Depend on Figure 3 It can be seen that the α-amylase inhibitory activity of peptide 31 (FYPQLFR) showed a concentration-dependent relationship. The IC of FYPQLFR was obtained by calculation. 50 The value is 2.54 mg / mL.

[0093] According to the inhibition rate results, the concentration IC of goat milk casein hydrolysates No. 1-3 was selected for in vitro inhibition of α-amylase experiment 50 The final concentrations of goat milk casein hydrolysates No. 1-3 were set to 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, and then the α-amylase inhibition experiment was carried out. The specific results can be seen in Figure 4 , Figure 5 and Figure 6 ,in Figure 4 It is the IC of goat milk casein enzymatic hydrolysate No. 1 50 Value result graph, Figure 5 It is the IC of goat milk casein enzymatic hydrolysate No. 2 50 Value result graph, Figure 6 It is the IC of goat milk casein enzymatic hydrolysate No. 3 50 Value result graph.

[0094] Depend on Figure 4 , Figure 5 and Figure 6 It can be seen that the α-amylase 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. 50The values ​​were 0.17 mg / mL, 0.16 mg / mL and 0.34 mg / mL respectively. Compared with the candidate functional peptides, the IC 50 The value increased significantly, mainly because the relative content of the functional peptides in the goat casein hydrolysate was relatively stable but not high overall. Subsequently, the goat casein hydrolysates 1-4 were detected and analyzed by liquid chromatography-mass spectrometry, and the mass contents of 9 candidate peptides in goat casein hydrolysates 1-4 were obtained. The specific content can be seen in Table 5. It can be found that the relative content of FYPQLFR in goat casein hydrolysates 1-4 is 0.08% or more, which plays an important role in the hypoglycemic activity of goat casein hydrolysates.

[0095] Table 4

[0096]

[0097] Table 5

[0098]

[0099] 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 functional peptide composition having a hypoglycemic effect, characterized in that: The functional peptide composition comprises peptide segment FYPQLFR, peptide segment FFY, peptide segment LPW and peptide segment AWPQYLK.

2. A goat milk casein enzymatic hydrolysate, characterized in that: The goat milk casein enzymatic hydrolysate comprises the functional peptide composition according to claim 1; The preparation method of the goat casein enzymatic hydrolysate comprises: mixing 5 kg of goat casein raw material and 50 kg of pure water, adding 3 mol / L of food-grade sodium hydroxide solution at a stirring speed of 100 rpm, heating to 40°C and adjusting the pH to 8.0, and then adding 25 g of trypsin with an enzyme activity of 4000 U / g to perform enzymatic hydrolysis for 2.5 hours, during which stirring is maintained and sodium hydroxide solution is continuously added to maintain the pH value of the solution at 7.9-8.1; after the enzymatic hydrolysis is completed, the temperature is immediately raised to 90°C and kept warm for 20 minutes to perform enzyme inactivation treatment; after the temperature drops to 50°C, filtering, concentrating at 65°C, and spray drying to obtain the goat casein enzymatic hydrolysate; based on the mass of the goat casein enzymatic hydrolysate, the content of the peptide segment FYPQLFR is 0.26%, the content of the peptide segment FFY is 0.01%, the content of the peptide segment LPW is 0.94%, and the content of the peptide segment AWPQYLK is 0.21%; Or, the preparation method of the goat casein enzymatic hydrolysate comprises: mixing 20 kg of goat casein raw material and 160 kg of pure water, adding 3 mol / L of food-grade sodium hydroxide solution at a stirring speed of 100 rpm, heating to 45°C and adjusting the pH to 7.5, and then adding trypsin with an enzyme activity of 4000 U / g and a mass of 20 g to perform a first enzymolysis for 2.0 h, during which stirring is maintained and sodium hydroxide solution is continuously added to maintain the pH value of the solution at 7.4-7.6; after the first enzymolysis is completed, adding 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, maintaining stirring and a pH value of 7.4-7.6 for a second enzymolysis for 3.0 h; after the second enzymolysis is completed, the temperature is immediately raised to 90°C and kept warm for 30 min, and perform enzyme inactivation treatment; after the temperature is reduced to 50°C, filter, concentrate at 65°C, and spray-dry to obtain a goat casein enzymatic hydrolysate; based on the mass of the goat casein enzymatic hydrolysate, the content of the peptide segment FYPQLFR is 0.27%, the content of the peptide segment FFY is 0.02%, the content of the peptide segment LPW is 0.97%, and the content of the peptide segment AWPQYLK is 0.16%.

3. The goat milk casein enzymatic hydrolysate according to claim 2, 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%.

4. The method for preparing the goat milk casein enzymatic hydrolysate according to claim 2 or 3, characterized in that: include: 5 kg of goat milk casein raw material and 50 kg of pure water were mixed, 3 mol / L of 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, and then 25 g of trypsin with an enzyme activity of 4000 U / g was added thereto for enzymolysis for 2.5 hours, during which 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 minutes to inactivate the enzyme; After the temperature drops to 50°C, the mixture is filtered, concentrated at 65°C, and spray-dried to obtain goat milk casein enzymatic hydrolysate; Or, comprising: mixing 20 kg of goat milk casein raw material and 160 kg of pure water, adding 3 mol / L food-grade sodium hydroxide solution at a stirring speed of 100 rpm, heating to 45°C and adjusting the pH to 7.5, and then adding trypsin with an enzyme activity of 4000 U / g and a mass of 20 g to perform a first enzymolysis for 2.0 h, stirring is maintained and sodium hydroxide solution is continuously added to maintain the pH value of the solution at 7.4-7.6; after the first enzymolysis is completed, adding 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 to perform a second enzymolysis for 3.0 h while stirring and the pH value is 7.4-7.6; after the second enzymolysis is completed, immediately heating to 90°C and keeping the temperature for 30 min to perform enzyme inactivation treatment; filtering after the temperature drops to 50°C, concentrating at 65°C, and spray drying to obtain a goat milk casein enzymolysate.

5. Use of the functional peptide composition according to claim 1, the goat casein enzymatic hydrolysate according to claim 2 or 3, or the goat casein enzymatic hydrolysate prepared by the preparation method according to claim 4 in the preparation of hypoglycemic drugs.

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