Millet alcohol-soluble protein active peptide, functional peptide and preparation method and application thereof

Abstract

The present invention provides a millet alcohol-soluble protein active peptide, a functional peptide and a preparation method and application thereof, belonging to the technical field of functional polypeptides. The present invention uses defatted millet as raw material and extracts it with an ethanol aqueous solution. The obtained alcohol-soluble protein is subjected to a dual treatment of ultrasonic crushing and enzymatic hydrolysis to obtain a millet alcohol-soluble protein active peptide with a blood sugar-lowering and fat-reducing effect. The three functional peptides are further separated and purified. The active peptide and the functional peptide both have strong α-amylase and α-glucosidase inhibitory activity, and can significantly improve the fasting blood sugar level and lipid metabolism disorder of diabetic mice. They can be used alone or as an active ingredient in the preparation of medicines, foods or health products.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional polypeptides, and in particular relates to a millet alcohol-soluble protein active peptide, a functional peptide, and a preparation method and application thereof. Background Art

[0002] Diabetes is a metabolic disease characterized by elevated blood sugar levels caused by impaired insulin metabolism or insufficient secretion. Hyperglycemia caused by diabetes can also disrupt lipid metabolism, leading to dyslipidemia and an increased risk of other diseases. Therefore, preventing and treating diabetes and its complications is urgent.

[0003] The main source of blood sugar in the human body is the diet, and the main components of carbohydrates in the diet are starch and sugars. Starch and sugars have a large molecular weight and cannot be directly absorbed by the human body and enter the blood circulation. α-amylase and α-glucosidase are important enzymes in the intestinal digestion and absorption process, which can hydrolyze starch and sugars into easily absorbed monosaccharides (glucose and fructose). Therefore, the source of blood sugar can be reduced by inhibiting the activity of α-amylase and α-glucosidase. At present, the inhibitors approved for clinical use include acarbose, voglibose and miglitol, but these drugs cannot effectively control complications and also produce some side effects of gastrointestinal discomfort.

[0004] Bioactive peptides are a class of protein fragments with specific physiological effects, composed of two or more amino acid residues linked by peptide bonds. Bioactive peptides have diverse functions, and those with hypoglycemic properties are often used in the development of drugs to prevent and treat diabetes and its complications. Compared to clinically approved inhibitor drugs, α-amylase and α-glucosidase inhibitory peptides offer the advantages of significant enzyme inhibition and fewer toxic side effects. However, current separation and purification technologies for α-amylase and α-glucosidase inhibitory peptides are complex, resulting in low product yields and high prices, making them unsuitable for industrial-scale production. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a millet alcohol-soluble protein active peptide, which has the effect of lowering blood sugar and reducing fat, can be used as a new type of α-amylase and α-glucosidase inhibitory peptide, effectively regulate the fasting blood sugar level and lipid metabolism disorders of diabetic patients, and provide a new means for the prevention and treatment of diabetes and its complications.

[0006] The present invention provides a millet alcohol-soluble protein active peptide, which is prepared by the following steps:

[0007] Extracting defatted millet with 60% to 80% ethanol aqueous solution to obtain millet alcohol-soluble protein;

[0008] The millet alcohol-soluble protein is crushed and then hydrolyzed with protease, and the hydrolyzate is collected as the millet alcohol-soluble protein active peptide.

[0009] Preferably, the mass ratio of the defatted millet to the ethanol aqueous solution is 1:5-10.

[0010] Preferably, the extraction temperature is 48-52°C;

[0011] The extraction time is 1 to 3 hours.

[0012] Preferably, after the extraction, the method further comprises dialyzing the extract and collecting the solution in the dialysis bag;

[0013] The molecular weight cut-off of the dialysis bag is 6000-8000 Da.

[0014] Preferably, the protease comprises at least one of the following enzymes: alkaline protease, pepsin, trypsin, flavor protease and neutral protease.

[0015] Preferably, the pH value of the system during the enzymatic hydrolysis is 2 to 8;

[0016] The temperature of the enzymatic hydrolysis is 30-60°C;

[0017] The enzymatic hydrolysis time is 1 to 5 hours;

[0018] During the enzymolysis, the added mass of the protease accounts for 1% to 3% of the mass of the millet.

[0019] Preferably, the crushing method is ultrasonic crushing;

[0020] During the ultrasonic crushing, the power of the ultrasonic wave is 100 to 500 W, and the time of the ultrasonic crushing is 5 to 25 minutes.

[0021] The present invention provides a functional peptide separated from the millet alcohol-soluble protein active peptide, wherein the functional peptide is one or more of the polypeptides having amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 3.

[0022] The present invention provides a method for separating the functional peptide, comprising the following steps:

[0023] dissolving the millet alcohol-soluble protein active peptide and filtering it with a filter membrane, collecting the filtrate and purifying it through reverse-phase high performance liquid chromatography, and collecting the fraction with higher inhibitory activity against α-amylase and α-glucosidase as the functional peptide;

[0024] The conditions for the reversed-phase high performance liquid chromatography purification are:

[0025] A C18 chromatographic column was used, and the eluent was mobile phase A and / or mobile phase B; the mobile phase A was an aqueous solution containing 0.1% by volume of formic acid, and the mobile phase B was an acetonitrile aqueous solution containing 0.1% by volume of formic acid, with a flow rate of 250 nL / min;

[0026] The liquid phase gradient program was as follows: 0–50 min, 4%–50% volume percentage of mobile phase B;

[0027] 50-54 min, 50%-100% mobile phase B by volume;

[0028] 54-60 min, 100% by volume of mobile phase B;

[0029] The liquid chromatography column was equilibrated with mobile phase A having a volume percentage of 95%.

[0030] The present invention provides use of the millet prolamin active peptide or the functional peptide in preparing a medicine for preventing and / or treating diabetes and its complications.

[0031] The present invention provides a millet alcohol-soluble protein active peptide, which is prepared by the following steps: extracting defatted millet with a 60% to 80% ethanol aqueous solution by volume to obtain millet alcohol-soluble protein; crushing the millet alcohol-soluble protein and performing protease hydrolysis, and collecting the enzymatic hydrolysis solution as the millet alcohol-soluble protein active peptide. The millet alcohol-soluble protein active peptide provided by the present invention has the following advantages:

[0032] (1) The present invention uses millet as a raw material to prepare millet alcohol-soluble protein active peptides with blood sugar and fat-reducing effects. The raw materials used are cheap, which reduces the production cost of the active peptides;

[0033] (2) The present invention uses 60% to 80% by volume ethanol aqueous solution extraction combined with enzymatic hydrolysis technology to prepare millet alcohol-soluble protein active peptides with blood sugar and fat-reducing effects. The preparation process is simple to operate and the protein purity is above 95%;

[0034] (3) The millet alcohol-soluble protein active peptide with blood sugar and fat-reducing effects prepared by the present invention is a new type of α-amylase and α-glucosidase inhibitory peptide for treating or preventing diabetes, and can effectively regulate the fasting blood sugar level and lipid metabolism disorder of diabetic mice.

[0035] Furthermore, the millet alcohol-soluble protein active peptide provided by the present invention specifically limits ultrasonic crushing during crushing, which is beneficial to unfolding the structure of the protein, so that the active peptide obtained after enzymatic hydrolysis has the characteristics of small molecular weight, rich content of specific amino acids and strong enzyme inhibitory activity.

[0036] Furthermore, the millet alcohol-soluble protein active peptide provided by the present invention specifically limits the type of protease, among which the millet alcohol-soluble protein active peptide obtained by alkaline protease hydrolysis has the highest inhibition rate on α-amylase and α-glucosidase (30.49% and 20.83% respectively), followed by trypsin.

[0037] The present invention also provides a functional peptide isolated from the millet alcohol-soluble protein active peptide, wherein the amino acid sequence of the functional peptide is one or more of SEQ ID NO: 1 to SEQ ID NO: 3. Compared with the active peptide, the functional peptide isolated by the present invention has stronger α-amylase and α-glucosidase inhibitory activity. The inhibition rates of SPALLLIPF, ILPFFGR, and QLPFNPLR on α-amylase are 63.16%, 70.54%, and 74.27%, respectively, and the inhibition rates on α-glucosidase are 79.26%, 86.47%, and 83.16%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results of optimization of millet alcohol-soluble protein extraction are shown in Table 1. Different lowercase letters indicate significant differences (P<0.05).

[0039] Figure 2 The results of the effect of the millet alcohol-soluble protein active peptide prepared by the present invention on the blood glucose content of diabetic mice, * indicates a significant difference compared with the normal group (P < 0.05), and # indicates a significant difference compared with the model group (P < 0.05);

[0040] Figure 3 These are the mass spectra of the three highly active α-amylase and α-glucosidase inhibitory peptides screened in Example 14, where A is the mass spectrum of the functional peptide SPALLLIPF, B is the mass spectrum of the functional peptide ILPFFGR, and C is the mass spectrum of the functional peptide QLPFNPLR. DETAILED DESCRIPTION

[0041] The present invention provides a millet alcohol-soluble protein active peptide, which is prepared by the following steps:

[0042] Extracting defatted millet with 60% to 80% ethanol aqueous solution to obtain millet alcohol-soluble protein;

[0043] The millet alcohol-soluble protein is crushed and then hydrolyzed with protease, and the hydrolyzate is collected as the millet alcohol-soluble protein active peptide.

[0044] The invention extracts defatted millet with 60% to 80% ethanol aqueous solution by volume to obtain millet alcohol-soluble protein.

[0045] In the present invention, the method for preparing the defatted millet is preferably to extract the millet with n-hexane and collect the precipitate. During the n-hexane extraction, the solid-liquid ratio is preferably 1:5-10. The n-hexane extraction is preferably carried out under water bath conditions. The temperature of the n-hexane extraction is preferably 35-39°C, more preferably 37°C. The time of the n-hexane extraction is preferably 3-5 hours, most preferably 4 hours. After the n-hexane extraction, it is preferably allowed to stand for 2 hours. After the standing, the precipitate is separated and then ventilated and dried to remove the solvent. In order to improve the defatting effect, it is preferred to crush the millet and collect 50-100 mesh powder for processing. The defatting treatment is beneficial to remove the fat-soluble components present in the millet and reduce the impurity content in the subsequent ethanol-water extraction process.

[0046] In the present invention, the extraction temperature is preferably 48-52°C, more preferably 50°C. The extraction time is preferably 1-3 hours, more preferably 2 hours. The volume percentage of the ethanol-water solution is preferably 70%. The ethanol-water extraction is preferably performed in a water bath. During the ethanol-water extraction, the mass ratio of defatted millet flour to ethanol-water solution is preferably 1:5-10, more preferably 1:7. After the extraction, to further remove impurities, the extract is preferably centrifuged, the supernatant is collected, and dialyzed, with the solution in the dialysis bag collected. The centrifugation conditions are preferably 4°C at 5000-10000 rpm for 10-25 minutes, more preferably 4°C at 8000 rpm for 15 minutes. The molecular weight cut-off of the dialysis bag is preferably 6000-8000 Da, more preferably 7000 Da. After dialysis, the collected dialysate is preferably freeze-dried. The freeze-drying temperature is preferably -40--60°C, more preferably -50°C, and the time is preferably 24-72 hours, more preferably 48 hours. The vacuum degree is preferably 5 to 20 Pa, more preferably 10 to 15 Pa.

[0047] After obtaining the millet alcohol-soluble protein, the invention crushes the millet alcohol-soluble protein and performs enzymatic hydrolysis with protease, and collects the enzymatic hydrolysis liquid as the millet alcohol-soluble protein active peptide.

[0048] In the present invention, the crushing method is preferably ultrasonic crushing. During the ultrasonic crushing, the ultrasonic power is preferably 100-500W, more preferably 200-400W, and most preferably 300W; the ultrasonic crushing time is preferably 5-25 minutes, more preferably 10-20 minutes, and most preferably 15 minutes.

[0049] In the present invention, the protease preferably comprises at least one of the following: alkaline protease, pepsin, trypsin, flavor protease, or neutral protease, with alkaline protease and trypsin being more preferred. Experiments have shown that different types of proteases have different inhibitory activities on the final product of enzymatic hydrolysis. The preparation process of alkaline protease is most advantageous for obtaining active peptides with high inhibition rates against α-amylase and α-glucosidase. The pH of the system during enzymatic hydrolysis is preferably between 2 and 8, with the pH value determined based on the hydrolysis conditions of the different types of proteases. The enzymatic hydrolysis temperature is preferably between 30 and 60°C, more preferably between 37 and 55°C, and most preferably at 50°C. The enzymatic hydrolysis time is preferably between 1 and 2 hours, more preferably 1.5 hours. The amount of protease added during enzymatic hydrolysis is preferably between 1% and 3% of the mass of the millet, more preferably between 2% and 3%. After enzymatic hydrolysis, an enzyme inactivation and centrifugation step is preferably performed to completely inactivate the protease in the system. The enzyme inactivation method preferably involves inactivating the hydrolysis solution at 100°C for 10 to 20 minutes. The centrifugation condition is preferably 4°C at 5000-10000 rpm for 10-25 min, more preferably 4°C at 8000 rpm for 15 min.

[0050] In the present invention, after centrifugation, the supernatant is collected and freeze-dried. The freeze-drying temperature is preferably -40 to -60°C, more preferably -50°C; the drying time is preferably 24 to 72 hours, more preferably 48 hours. The vacuum degree is preferably 5 to 20 Pa, more preferably 10 to 15 Pa.

[0051] In the present invention, the effects of the prepared millet prolamin active peptides on the inhibition rate of α-amylase and α-glucosidase were measured respectively. The results showed that the millet prolamin active peptides prepared by the present invention all had α-amylase and α-glucosidase inhibitory activity, but the activities of the millet prolamin active peptides prepared by different proteases were different. The inhibitory activity was ranked from high to low as alkaline protease > trypsin > pepsin > neutral protease > flavor protease. The experiment showed that the millet prolamin peptides that were not enzymatically hydrolyzed but only ultrasonically fragmented had very low α-amylase and α-glucosidase inhibitory activity. At the same time, the prepared millet prolamin active peptides were administered to diabetic model mice. The results showed that the intervention of the millet prolamin active peptides could effectively improve the blood sugar levels of the model mice and improve the glucose tolerance of the model mice. At the same time, after the intervention of the millet prolamin active peptides, the total cholesterol, triglyceride, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol levels in the intervention group were significantly lower than those in the model group, indicating that the millet prolamin active peptides have the effect of improving hyperlipidemia in diabetic mice. This shows that the millet alcohol-soluble protein active peptide provided by the present invention has the effect of lowering blood sugar and reducing fat, and provides a basis for the prevention and treatment of diabetes and its complications.

[0052] The present invention provides a functional peptide separated from the millet alcohol-soluble protein active peptide, wherein the functional peptide is one or more of the polypeptides with amino acid sequences as shown in SEQ ID NO: 1 (SPALLLIPF), SEQ ID NO: 2 (ILPFFGR) and SEQ ID NO: 3 (QLPFNPLR).

[0053] The invention provides a method for separating the functional peptides, comprising the following steps: dissolving the millet alcohol-soluble protein active peptide, filtering the mixture with a filter membrane, collecting the filtrate, purifying the mixture through reverse-phase high-performance liquid chromatography, and collecting a fraction with high inhibitory activity against α-amylase and α-glucosidase as the functional peptide; the conditions for the reverse-phase high-performance liquid chromatography purification are: using a C18 chromatographic column, and the eluent is a mobile phase A and / or a mobile phase B; the mobile phase A is an aqueous solution containing 0.1% by volume of formic acid, and the mobile phase B is an acetonitrile aqueous solution containing 0.1% by volume of formic acid, with a flow rate of 250 nL / min; the liquid phase gradient program is as follows: 0-50 min, mobile phase B with a volume percentage of 4-50%; 50-54 min, mobile phase B with a volume percentage of 50-100%; 54-60 min, mobile phase B with a volume percentage of 100%; and the liquid chromatography column is equilibrated with a mobile phase A with a volume percentage of 95%. The purified millet alcohol-soluble protein active peptides were detected by liquid chromatography-tandem mass spectrometry to obtain three functional peptides.

[0054] The present invention uses liquid chromatography tandem mass spectrometry to determine the functional peptides of the purified millet alcohol-soluble protein, and combines with the PeptideRanker toolkit (http: / / distilldeep.ucd.ie / PeptideRanker / ) to screen out three peptides with a bioactivity score higher than 0.8 and a confidence level greater than 30, namely SPALLLIPF, ILPFFGR, and QLPFNPLR.

[0055] The present invention artificially synthesized three peptides and then performed functional verification. Compared with the active peptides, the inhibition rates of SPALLLIPF, ILPFFGR, and QLPFNPLR functional peptides on α-amylase were 63.16%, 70.54%, and 74.27%, respectively, and the inhibition rates on α-glucosidase were 79.26%, 86.47%, and 83.16%, respectively.

[0056] The following is a detailed description of a millet alcohol-soluble protein active peptide, a functional peptide, a preparation method and an application thereof provided by the present invention in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] Extraction method of millet alcohol-soluble protein

[0059] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0060] (2) Extraction: Defatted millet flour was mixed with 60% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialysate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein.

[0061] Example 2

[0062] Extraction method of millet alcohol-soluble protein

[0063] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:7, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0064] (2) Extraction: Defatted millet flour was mixed with 60% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialysate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein.

[0065] Example 3

[0066] Extraction method of millet alcohol-soluble protein

[0067] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:10, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0068] (2) Extraction: Defatted millet flour was mixed with 60% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialysate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein.

[0069] Example 4

[0070] Extraction method of millet alcohol-soluble protein

[0071] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0072] (2) Extraction: Defatted millet flour was mixed with 70% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialyzate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein.

[0073] Example 5

[0074] Extraction method of millet alcohol-soluble protein

[0075] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0076] (2) Extraction: Defatted millet flour was mixed with 80% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialyzate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein.

[0077] Example 6

[0078] The millet alcohol-soluble proteins prepared in Examples 1 to 5 were tested for purity.

[0079] Protein content determination: According to GB / T 5009.5-2016, the content of millet alcohol-soluble protein was determined by Kjeldahl method.

[0080] The results are as follows Figure 1 As shown in the results, the ratio of n-hexane has little effect on the purity of millet alcohol-soluble protein. As the ratio of n-hexane increases, the content of millet alcohol-soluble protein obtained by extraction remains basically unchanged. The volume percentage of ethanol has a greater impact on the purity of millet alcohol-soluble protein. When 70% by volume ethanol is used for extraction, the millet alcohol-soluble protein content is the highest.

[0081] Example 7

[0082] Preparation method of millet alcohol-soluble protein active peptide

[0083] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0084] (2) Extraction: Defatted millet flour was mixed with 70% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialyzate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein;

[0085] (3) Enzymatic hydrolysis: The millet alcohol-soluble protein was dissolved into a 10 mg / mL suspension, and the suspension was treated with ultrasonic cell disruption technology at 400 W for 20 min. The pH of the solution was adjusted to 8 and the temperature to 50°C. Alkaline protease with a substrate concentration of 3% was added for enzymatic hydrolysis for 2 h. The enzymatic hydrolyzate was inactivated at 100°C for 10 min, and the supernatant was centrifuged at 8000 rpm for 15 min at 4°C to obtain the supernatant, which was freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain the millet alcohol-soluble protein active peptide.

[0086] Example 8

[0087] Preparation method of millet alcohol-soluble protein active peptide

[0088] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0089] (2) Extraction: Defatted millet flour was mixed with 70% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialyzate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein;

[0090] (3) Enzymatic hydrolysis: The millet alcohol-soluble protein was dissolved into a 10 mg / mL suspension, and the suspension was treated with ultrasonic cell disruption technology at 400 W for 20 min. The pH of the solution was adjusted to 2 and the temperature to 37°C. Pepsin with a substrate concentration of 3% was added for enzymatic hydrolysis for 2 h. The enzymatic hydrolyzate was inactivated at 100°C for 10 min, and the supernatant was centrifuged at 8000 rpm for 15 min at 4°C to obtain the supernatant, which was freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain the millet alcohol-soluble protein active peptide.

[0091] Example 9

[0092] Preparation method of millet alcohol-soluble protein active peptide

[0093] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0094] (2) Extraction: Defatted millet flour was mixed with 70% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialyzate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein;

[0095] (3) Enzymatic hydrolysis: The millet alcohol-soluble protein was dissolved into a 10 mg / mL suspension, and the suspension was treated with ultrasonic cell disruption technology at 400 W for 20 min. The pH of the solution was adjusted to 8 and the temperature to 37°C. Trypsin with a substrate concentration of 3% was added for enzymatic hydrolysis for 2 h. The enzymatic hydrolyzate was inactivated at 100°C for 10 min, and the supernatant was centrifuged at 8000 rpm for 15 min at 4°C to obtain the supernatant, which was freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain the millet alcohol-soluble protein active peptide.

[0096] Example 10

[0097] Preparation method of millet alcohol-soluble protein active peptide

[0098] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0099] (2) Extraction: Defatted millet flour was mixed with 70% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialyzate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein;

[0100] (3) Enzymatic hydrolysis: The millet alcohol-soluble protein was dissolved into a 10 mg / mL suspension, and the suspension was treated with ultrasonic cell disruption technology at 400 W for 20 min. The pH of the solution was adjusted to 7 and the temperature to 45°C. A neutral protease with a substrate concentration of 3% was added for enzymatic hydrolysis for 2 h. The enzymatic hydrolyzate was inactivated at 100°C for 10 min, and the supernatant was centrifuged at 8000 rpm for 15 min at 4°C. The supernatant was taken and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain the millet alcohol-soluble protein active peptide with hypoglycemic and lipid-reducing effects.

[0101] Example 11

[0102] Preparation method of millet alcohol-soluble protein active peptide

[0103] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0104] (2) Extraction: Defatted millet flour was mixed with 70% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialyzate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein;

[0105] (3) Enzymatic hydrolysis: The millet alcohol-soluble protein was dissolved into a 10 mg / mL suspension, and the suspension was treated with ultrasonic cell disruption technology at 400 W for 20 min. The pH of the solution was adjusted to 7 and the temperature to 50°C. Flavor protease with a substrate concentration of 3% was added for enzymatic hydrolysis for 2 h. The enzymatic hydrolyzate was inactivated at 100°C for 10 min, and the supernatant was centrifuged at 8000 rpm for 15 min at 4°C. The supernatant was taken and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein active peptides with hypoglycemic and lipid-reducing effects.

[0106] Comparative Example 1

[0107] Preparation method of millet alcohol-soluble protein active peptide

[0108] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0109] (2) Extraction: Defatted millet flour was mixed with 70% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialyzate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein;

[0110] (3) Enzymatic hydrolysis: The millet alcohol-soluble protein was dissolved into a 10 mg / mL suspension, and the suspension was treated with ultrasonic cell disruption technology at 400 W for 20 min. The supernatant was centrifuged at 8000 rpm for 15 min at 4 ° C and freeze-dried (vacuum degree 20 Pa, temperature -50 ° C, time 48 h) to obtain the millet alcohol-soluble protein active peptide with hypoglycemic and lipid-reducing effects.

[0111] Example 12

[0112] The millet alcohol-soluble protein active peptides with hypoglycemic and fat-reducing effects prepared in Examples 7 to 11 and Comparative Example 1 were functionally tested.

[0113] Determination of degree of hydrolysis: Pipette 400 μL of millet alcohol-soluble protein hydrolysate into a test tube containing 3 mL of OPA, shake and mix thoroughly, and react for 2 minutes. Measure the absorbance at 340 nm. Draw a standard curve using serine as the standard. Calculate the degree of hydrolysis according to Equation 1.

[0114]

[0115] Where: h represents the number of millimoles of peptide bonds cleaved per gram of protein in the protease hydrolysate (meq / g protein), and htot represents the number of millimoles of total peptide bonds per gram of raw protein.

[0116] α-Amylase Inhibition Rate Determination: Add 100 μL of millet alcohol-soluble peptide to a test tube containing 500 μL of α-amylase solution and incubate in a 37°C water bath for 10 minutes. Then, add 500 μL of 1% starch solution and incubate in a 37°C water bath for 10 minutes. Then, add 1 mL of DNS colorimetric reagent and incubate in a boiling water bath for 5 minutes. Measure the absorbance at 540 nm and calculate the α-amylase inhibition rate according to Equation II.

[0117]

[0118] Wherein: A1 is the sample group, A2 is the background group without adding DNS, and A3 is the blank group without adding sample.

[0119] α-glucosidase inhibition rate determination: Pipette 25 μL of millet prolamin peptide into a test tube containing 50 μL of α-glucosidase solution and incubate in a 37°C water bath for 10 minutes. Then, add 25 μL of PNPG solution and incubate in a 37°C water bath for 5 minutes. Measure the absorbance at 405 nm, and calculate the α-glucosidase inhibition rate according to Equation III.

[0120]

[0121] Wherein: A1 is the sample group, A2 is the background group without PNPG, and A3 is the blank group without sample.

[0122] Table 1 Hydrolysis degree of millet alcohol-soluble protein, α-amylase and α-glucosidase inhibition rate of millet alcohol-soluble protein active peptides in Examples 7 to 11 and Comparative Example 1

[0123]

[0124]

[0125] Note: “—” represents not detected; different lowercase letters in the same column represent significant differences (P<0.05).

[0126] As shown in Table 1, compared with Comparative Example 1, the protease treatment in Examples 7 to 11 significantly improved the hydrolysis degree of millet alcohol-soluble protein and the enzyme inhibition rate of millet alcohol-soluble protein active peptides. Among them, alkaline protease had the best hydrolysis effect, and the obtained millet alcohol-soluble protein active peptides had the highest inhibition rate on α-amylase and α-glucosidase.

[0127] Example 13

[0128] Effects of Millet Prolamin Active Peptides on Diabetic Mice

[0129] 36 4-week-old C57BL / 6J male mice were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. After 1 week of adaptive feeding, they were randomly divided into groups according to their body weight. 12 mice were fed a normal diet as a normal control group. The remaining mice were continuously fed a high-fat diet for 12 weeks. At the 5th week, the high-fat-fed mice were fasted for 12 hours and intraperitoneally injected with 100 mg / kg STZ to establish a type II diabetes model. The successfully modeled diabetic mice were divided into a model group and an intervention group. The intervention group was gavaged with millet alcohol-soluble protein active peptide (prepared by the method of Example 7) every day, and the model group was gavaged with an equal amount of normal saline every day. After continuous gavage for 4 weeks, the mice were fasted for 12 hours and gavaged with glucose solution at a dose of 1 g / kg. Blood glucose was then measured using a blood glucose meter at 0, 15 min, 30 min, 60 min, 90 min, and 120 min after gavage. After 5 weeks of continuous gavage, blood was collected from the orbits and the mice were killed by dislocation. The serum total cholesterol, triglycerides, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol were determined by kit method.

[0130] Table 2 Serum parameters of mice in different treatment groups

[0131]

[0132]

[0133] Note: Different lowercase letters in the same row represent significant differences (P<0.05).

[0134] like Figure 2 As shown in the results, compared with the normal group, the blood glucose levels of the model group mice increased sharply after glucose injection, indicating that their glucose tolerance was significantly impaired. The blood glucose levels of the intervention group mice were significantly improved compared with the model group, indicating that the millet alcohol-soluble protein active peptide has the effect of improving glucose tolerance in diabetic mice.

[0135] As shown in Table 2, compared with the normal group, the model group mice had higher levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. After intervention with millet prolamin active peptides, the total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol levels in the intervention group were significantly lower than those in the model group, indicating that millet prolamin active peptides have the effect of improving hyperlipidemia in diabetic mice.

[0136] Example 14

[0137] Isolation, purification and enzyme inhibitory activity of millet alcohol-soluble peptides with hypoglycemic and lipid-reducing effects

[0138] (1) Pretreatment: Wash and dry the millet, grind it with a grinder, pass it through a 60-mesh sieve, mix it with n-hexane solution at a mass ratio of 1:5, shake it in a water bath at 37°C for 4 h, let it stand at room temperature for 2 h, collect the precipitate and dry it in a fume hood for 12 h to obtain defatted millet powder;

[0139] (2) Extraction: Defatted millet flour was mixed with 70% ethanol solution in a mass ratio of 1:7, shaken in a 50°C water bath for 2 h, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was dialyzed in a dialysis bag for 12 h and repeated 3 times. The dialyzate was collected and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain millet alcohol-soluble protein;

[0140] (3) Enzymatic hydrolysis: The millet alcohol-soluble protein was dissolved into a 10 mg / mL suspension, and the suspension was treated with ultrasonic cell disruption technology at 400 W for 20 min. The pH of the solution was adjusted to 8 and the temperature to 50°C. Alkaline protease with a substrate concentration of 3% was added for enzymatic hydrolysis for 2 h. The enzymatic hydrolyzate was inactivated at 100°C for 10 min, and the supernatant was centrifuged at 8000 rpm for 15 min at 4°C. The supernatant was taken and freeze-dried (vacuum degree 20 Pa, temperature -50°C, time 48 h) to obtain the millet alcohol-soluble protein active peptide with hypoglycemic and lipid-reducing effects.

[0141] (4) Purification: The active peptide of millet alcohol-soluble protein was filtered through a 3 kDa ultrafiltration membrane, and the fractions with higher α-amylase and α-glucosidase inhibitory activities were collected and purified by reverse-phase high performance liquid chromatography. The purification conditions were as follows: a C18 chromatographic column was used, with an aqueous solution containing 0.1% formic acid as the mobile phase A, and an acetonitrile aqueous solution containing 0.1% formic acid as the mobile phase B. The flow rate was 250 nL / min, and the liquid phase gradient was set as follows: 0-50 min, 4%-50% mobile phase B; 50-54 min, 50%-100% mobile phase B; 54-60 min, 100% mobile phase B.

[0142] like Figure 3 As shown, functional peptides were determined by liquid chromatography-tandem mass spectrometry, and three peptides with bioactivity scores higher than 0.8 and confidence levels greater than 30 were screened out using the PeptideRanker toolkit (http: / / distilldeep.ucd.ie / PeptideRanker / ), namely SPALLLIPF, ILPFFGR, and QLPFNPLR.

[0143] The three peptides were artificially synthesized and their functions were verified, and the enzyme inhibition rate was determined according to the method of Example 12.

[0144] Compared with the active peptides, the inhibition rates of the three functional peptides SPALLLIPF, ILPFFGR, and QLPFNPLR on α-amylase were 63.16%, 70.54%, and 74.27%, respectively, and the inhibition rates on α-glucosidase were 79.26%, 86.47%, and 83.16%, respectively.

[0145] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A functional peptide separated from millet alcohol-soluble protein active peptide, characterized in that: The functional peptide is one or more of the polypeptides whose amino acid sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 3; The millet alcohol-soluble protein active peptide is prepared by the following steps: extracting defatted millet with 60% to 80% ethanol aqueous solution by volume to obtain millet alcohol-soluble protein; The millet alcohol-soluble protein is crushed and then enzymatically hydrolyzed with a protease, and the enzymatic hydrolyzate is collected as the millet alcohol-soluble protein active peptide; and functional peptides are separated from the millet alcohol-soluble protein active peptide; The mass ratio of the defatted millet to the ethanol aqueous solution is 1:5-10; The extraction temperature is 48-52°C; The extraction time is 1 to 3 hours; After the extraction, the extract is purified; The purification method is dialysis, and the solution in the dialysis bag is collected; The molecular weight cut-off of the dialysis bag is 6000-8000Da; The protease is alkaline protease; The pH value of the system during the enzymatic hydrolysis is 2-8; The temperature of the enzymatic hydrolysis is 30-60°C; The enzymatic hydrolysis time is 1 to 5 hours; The added mass of protease during the enzymatic hydrolysis accounts for 1% to 3% of the mass of the millet; The crushing method is ultrasonic crushing; During the ultrasonic crushing, the ultrasonic power is 100-500W, and the ultrasonic crushing time is 5-25min; The separation method comprises dissolving the millet alcohol-soluble protein active peptide and filtering it with a filter membrane, collecting the filtrate and purifying it through reverse-phase high-performance liquid chromatography, and collecting the fraction with high inhibitory activity against α-amylase and α-glucosidase as the functional peptide; the conditions for the reverse-phase high-performance liquid chromatography purification are as follows: using a C18 chromatographic column, the eluent is mobile phase A and / or mobile phase B; the mobile phase A is an aqueous solution containing 0.1% formic acid by volume, the mobile phase B is an acetonitrile aqueous solution containing 0.1% formic acid by volume, and the flow rate is 250 nL / min; the liquid phase gradient program is as follows: 0-50 min, mobile phase B with a volume percentage of 4%-50%; 50-54 min, mobile phase B with a volume percentage of 50%-100%; 54-60 min, mobile phase B with a volume percentage of 100%; the liquid chromatography column is equilibrated with mobile phase A with a volume percentage of 95%; The purified millet alcohol-soluble protein active peptide was determined by liquid chromatography-tandem mass spectrometry to obtain the functional peptide.

2. Use of the functional peptide according to claim 1 in the preparation of a drug for preventing and / or treating diabetes.

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