Selenium-rich black bean polypeptide and application thereof in preparation of hypoglycemic products

Abstract

According to the selenium-rich black bean polypeptide and the application thereof in preparation of the hypoglycemic product, compared with common black beans, the selenium-rich black bean polypeptide extracted from selenium-rich black beans has the advantages that the selenium-rich black bean polypeptide has a better inhibition effect on alpha-amylase; two novel selenium-rich black bean polypeptides Se-MetIP and TLTSe-MetLR are screened out from the selenium-rich black bean polypeptides, and the two novel selenium-rich black bean polypeptides have inhibitory activity on alpha-amylase; the selenium-rich black bean polypeptide with hypoglycemic activity has important significance for controlling alpha-amylase to reduce the source of glucose so as to prevent diabetes and high-valued utilization of selenium-rich black beans.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and in particular to a selenium-enriched black bean polypeptide and an application thereof in the preparation of a blood sugar-lowering product. Background Art

[0002] Diabetes mellitus (DM) is a metabolic disease divided into type 1 diabetes mellitus (T1DM, insulin-dependent) and type 2 diabetes mellitus (T2DM, non-insulin-dependent). After a meal, when blood glucose levels are high, insulin is released from pancreatic β cells, stimulating glucose uptake in muscle and adipose tissue and promoting glucose production in the liver; hyperglycemia occurs when insulin secretion cannot offset insulin resistance. Skeletal muscle, liver, and adipose tissue mainly show insulin resistance. Insulin resistance leads to impaired glucose uptake in skeletal muscle, suppressed glucose uptake in the liver, and subsequent increase in blood glucose levels. Lipolysis in adipocytes is disturbed, and the delivery of free fatty acids is enhanced. Fatty acids accumulate in skeletal muscle, liver, and pancreas, promoting insulin resistance, increasing hepatic glucose production, and impairing β-cell function.

[0003] Human blood sugar mainly comes from diet, and the main component of carbohydrates in diet is starch, which has a large molecular chain and cannot be directly absorbed into the human blood circulation. Instead, it needs to be converted into monosaccharides (glucose and fructose) through the synergistic action of amylase and various α-glucosidases on the villi of the small intestine before it can be absorbed. Therefore, the source of glucose can be reduced by controlling α-amylase.

[0004] Soybeans are rich in protein, with a content of up to 40%. They also contain lecithin and other effective ingredients that promote brain development, and have extremely high nutritional value and application value. Black beans, also known as black beans, are the black seeds of the leguminous plant soybean. They are used for yin deficiency, thirst, dizziness, sweating due to physical weakness, low back pain due to kidney deficiency, edema, oliguria, pain and cramps, numbness of hands and feet, and drug and food poisoning. Compared with ordinary beans, selenium-enriched beans have a high content of selenium. Selenium can participate in the synthesis and transformation of various substances in the body along the sulfur metabolism pathway to form different organic selenium compounds with high bioavailability. At present, the research on selenium-enriched beans mainly focuses on the research on artificial selenium enrichment, and the research on organic selenium in natural selenium-enriched beans is relatively lacking. Therefore, the study of organic selenium in selenium-enriched beans is conducive to the development of selenium resources and improves the quality and economic benefits of beans.

[0005] Bioactive peptides are specific protein fragments that have a positive impact on the physiological functions of biological organisms and human health. These peptides have the following characteristics: (1) high targeted biological activity; (2) low toxicity and reduced incidence of tissue aggregation in the body; (3) high structural diversity; and (4) small size (relative to antibodies). These properties allow peptides to be used as therapeutic agents with anti-diabetic, antibacterial, antioxidant, antithrombotic and antihypertensive functions in human health. Bioactive peptides extracted from plants or microorganisms can control blood sugar levels and reduce insulin resistance to fight diabetes. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a selenium-rich black bean polypeptide and its application in the preparation of hypoglycemic products. The selenium-rich black bean polypeptide with hypoglycemic activity is of great significance for controlling α-amylase to reduce the source of glucose to prevent diabetes and for the high-value utilization of selenium-rich black beans.

[0007] To achieve the above object, the present invention provides the following technical solution: a selenium-rich black bean polypeptide, which is obtained by enzymatically hydrolyzing selenium-rich black beans with alkaline protease and then freeze-drying the enzymatically hydrolyzed selenium-rich black beans.

[0008] Furthermore, the dosage of the alkaline protease is 1% to 5%.

[0009] The present invention also provides the use of the selenium-enriched black bean polypeptide in the preparation of α-amylase inhibitors.

[0010] Furthermore, the molecular weight of the selenium-rich black bean polypeptide is less than 1 kDa.

[0011] The present invention also provides the use of the selenium-rich black bean polypeptide in the preparation of a blood sugar-lowering product.

[0012] Furthermore, the molecular weight of the selenium-rich black bean polypeptide is less than 1 kDa.

[0013] The present invention also provides a product with blood sugar lowering function, wherein the active ingredient comprises the selenium-rich black bean polypeptide, and the concentration of the selenium-rich black bean polypeptide is 2 mg / mL to 10 mg / mL.

[0014] The present invention also provides a selenium-rich black bean blood sugar lowering peptide, which is screened from the above-mentioned selenium-rich black bean polypeptides and includes the selenium-rich black bean blood sugar lowering peptide Se-MetIP and / or the selenium-rich black bean blood sugar lowering peptide TLTSe-MetLR.

[0015] The present invention also provides the use of the selenium-rich black bean hypoglycemic peptide in the preparation of products with hypoglycemic function or α-amylase inhibitors.

[0016] The present invention also provides a product with blood sugar lowering function, wherein the active ingredient comprises the selenium-rich black bean blood sugar lowering peptide, and the concentration of the selenium-rich black bean blood sugar lowering peptide is 0.1 mg / mL to 4 mg / mL.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention utilizes selenium-rich black bean polypeptides extracted from selenium-rich black beans. Compared with ordinary black beans, the selenium-rich black bean polypeptides have a better inhibitory effect on α-amylase and can be used to prepare high-efficiency blood sugar lowering products. In addition, the present invention screens out two novel selenium-rich black bean polypeptides Se-MetIP and TLTSe-MetLR from the selenium-rich black bean polypeptides, which have inhibitory activity on α-amylase and can be used as blood sugar lowering active ingredients. Cell experiments verify that the two novel selenium-rich black bean polypeptides Se-MetIP and TLTSe-MetLR may exert their blood sugar lowering effect by increasing glycogen content, and clarify their potential blood sugar lowering mechanism, which is of great significance for preventing diabetes and high-value utilization of selenium-rich black beans. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of selenium-enriched black bean peptide concentration on the inhibitory effect of α-amylase; Figure 2-1 is the molecular weight distribution of selenium-rich black bean peptides; Figure 2-2 is the chain length of selenium-rich black bean peptide; Figure 3 The α-amylase inhibitory activity of selenium-enriched black bean glucose-lowering peptide; Figure 4 The circular dichroism spectra of the interaction between selenium-enriched black bean glucose-lowering peptides Se-MetIP and TLTSe-MetLR and α-amylase; Figure 5 The heat flow changes over time when the selenium-enriched black bean glucose-lowering peptides Se-MetIP and TLTSe-MetLR bind to α-amylase; Figure 6-1 It is the protein structure of α-amylase; Figure 6-2 The molecular structures of peptides Se-MetIP (a) and TLTSe-MetLR (b); Figure 6-3 Figure 5 shows the binding pattern of 5U3A protein and Se-MetIP peptide, where (a) is the two-dimensional binding pattern of peptide and protein, the green dotted line represents hydrogen bonding, and the red gear represents hydrophobic bonding; (b) is the position of the peptide in the three-dimensional structure of the protein (left); the three-dimensional binding pattern of the peptide and protein (right); Figure 6-4Figure 5 shows the binding pattern of 5U3A protein and TLTSe-MetLR peptide, where (a) is the two-dimensional binding pattern of peptide and protein, the green dotted line indicates hydrogen bonding, and the red gear indicates hydrophobic bonding; (b) is the position of the peptide in the three-dimensional structure of the protein (left); the three-dimensional binding pattern of the peptide and protein (right); Figure 7 The effect of selenium-enriched black bean glucose-lowering peptide on the survival rate of IR-HepG2 cells; Figure 8-1 The following are the effects of selenium-enriched black bean glucose-lowering peptide on glucose consumption in IR-HepG2 cells. ** indicates extremely significant difference compared with the blank control group (p<0.01); # indicates significant difference compared with the model control group (p<0.05); ## indicates extremely significant difference compared with the model control group (p<0.01).

[0019] Figure 8-2 This study is the effect of selenium-enriched black bean glucose-lowering peptide on glycogen synthesis in IR-HepG2 cell model. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0021] The present invention uses selenium-rich black bean protein polypeptide in the field of hypoglycemia, and the specific steps are as follows: Step 1: Extract selenium-rich black bean protein The selenium content of this black bean sample is 24.17μg / mg. Accurately weigh 100g of selenium-rich black bean powder, add 500mL of petroleum ether, stir magnetically for 2.5h, centrifuge at 4000r / min for 10min, take the lower precipitate and continue to defatting, repeat 3 times, and dry at room temperature for use. Take a certain amount of black soybean meal, add distilled water (pH value is 8.5) at 1:10 (m / V), stir in a 50℃ water bath for 2h, centrifuge at 4000r / min for 10min, take the supernatant, redissolve the precipitate, repeat the above operation, combine the two supernatants, adjust the supernatant with 1mol / LHCl solution (pH value is 4.5), centrifuge at 4000r / min for 10min, discard the supernatant, and retain the precipitate. Deionized water (pH 4.5) was added to the precipitate at a ratio of 1:10 (m / V), and the mixture was centrifuged at 4000 r / min for 10 min. The supernatant was discarded and the mixture was washed with water for 3 times. The precipitate was selenium-rich black bean protein isolate. A small amount of deionized water (pH 7) was added to the precipitate, and the selenium-rich black bean protein isolate was freeze-dried by vacuum freeze-drying, ground into powder, and sealed for storage.

[0022] Step 2: Preparation of selenium-enriched black bean peptide The selenium-enriched black bean protein prepared in step 1 was prepared into a solution with a substrate concentration of 5% (w / v) with deionized water, stirred thoroughly, kept warm in a boiling water bath for 15 minutes, and cooled to room temperature. 1% to 5% (enzyme / substrate) of alkaline protease was added to the selenium-enriched black bean protein solution, and the solution was adjusted to the optimal enzymolysis temperature and pH value for each enzyme, and the pH value was kept constant during the enzymolysis process. After the enzymolysis was completed, the enzyme was boiled and inactivated for 10 minutes to obtain a selenium-enriched black bean protein hydrolyzate; then centrifuged (4000r / min, 5min), the supernatant was taken, and vacuum freeze-dried to obtain selenium-enriched black bean polypeptide, which was ground into powder and sealed for storage.

[0023] Step 3: Determine the inhibition of α-amylase by the selenium-rich black bean peptide prepared in step 2 1) Preparation of sample solution and starch solution Sample solutions of different concentrations were prepared using 0.2 M sodium chloride phosphate buffer with a pH of 6.9 as solvent; 1% (w / v) starch solution; 2) Determination of the inhibition of α-amylase by the selenium-enriched black bean peptide prepared in step 2 Boil the starch solution in boiling water for 10 minutes to denature it, then cool to room temperature for later use.

[0024] Take 20 μL of sample solution and 10 μL of α-amylase (1 U / mL) and add them to a test tube. Incubate in a 37°C water bath for 15 min. Then add 500 μL of boiled and cooled starch solution. After reacting in a 37°C water bath for 15 min, add 600 μL of reaction stop solution (containing 1% DNS, 12% potassium sodium tartrate in 0.4 M Na 2 CO 3 Solution), boil for 15 min, cool to room temperature, pipette 200 μL of reaction solution into a 96-well plate, and measure the absorbance at 540 nm.

[0025] 3) According to the absorbance measured in step 2), the inhibition rate of selenium-rich black bean polypeptide on α-amylase is obtained, such as Figure 1 As shown, as the concentration of the peptide increases, the inhibition rate of α-amylase increases. In the concentration range of 2mg / mL to 10mg / mL, the inhibition rate of α-amylase increases with the concentration, from 37.55% to 61.17%, and the in vitro hypoglycemic activity of the selenium-rich black bean peptide is better than that of the ordinary black bean peptide.

[0026] Step 4: Separate and purify the selenium-rich black bean protein hydrolysate prepared in step 2 to obtain the selenium-rich black bean hypoglycemic peptide, and identify the structure of the selenium-rich black bean hypoglycemic peptide. The specific steps are as follows: 1) Separate the selenium-rich black bean protein hydrolysate using ultrafiltration membranes with molecular weight cutoffs of 10 kDa, 3 kDa and 1 kDa at room temperature, and the ultrafiltration pressure is 0.1 MPa. Figure 2-1 As shown in the figure, after ultrafiltration separation, the peptide content of the component with a molecular weight less than 1 kDa was significantly higher than that of the other two components, which indicates that the peptides in the selenium-enriched black bean hydrolysate are mainly short peptides with a molecular weight less than 1 kDa. 2) LC-MS / MS was used to identify the amino acid sequence of peptides in selenium-enriched black bean hydrolysate. The analysis results are shown in Figure 2-2 . Comparison and analysis with the database (uniprot-Sus-scrofa-122175-20220218) identified 509 peptide sequences from selenium-enriched black bean hydrolysate. These peptides are mainly short peptides with a molecular mass of less than 1 kDa, consisting of 3 to 9 amino acids.

[0027] Step 5. Screening and solid phase synthesis of selenium-enriched black bean hypoglycemic peptides The peptide sequences were scored by the PeptideRanker system. The higher the score (0-1), the higher the probability that the peptide had biological activity. The peptides that may have α-amylase inhibitory effect were compared in the BIOPEP-UWM database. The peptide sequences screened were solid-phase synthesized in the order from C-terminus to N-terminus using the in vitro Fmoc solid phase method. Dry crude peptides were prepared by amino acid connection reaction, peptide chain connection reaction and peptide chain shearing treatment. The crude peptides obtained by chemical synthesis were purified and detected by high-performance liquid chromatography combined with high-resolution mass spectrometry. The ion size of the target peptide was detected according to the mass-to-charge ratio, and the peak domain where the target peptide was located was determined. The samples with an analytical purity greater than 95% were freeze-dried to obtain the final pure peptide.

[0028] According to the amino acid composition characteristics of α-amylase inhibitory peptides, the present invention screened out peptide sequence 1 that may have α-amylase inhibitory activity from 509 peptide sequences: Se-MetIP, wherein Se-Met is selenomethionine; I is isoleucine; and P is proline; peptide sequence 2: TLTSe-MetLR, wherein T is threonine; L is leucine; Se-Met is selenomethionine, and R is arginine. The specific screening results are shown in Table 1.

[0029] Table 1 Chromatographic and mass spectrometric information of selenium-enriched black bean glucose-lowering peptides

[0030] Compared with BIOPEP-UWM data, the above two peptides have not been reported in research, indicating that the peptides screened by the present invention are novel. Therefore, these two novel peptides that may have α-amylase inhibitory activity were selected for further research.

[0031] Step 6: Detect the inhibition rate of α-amylase by selenium-rich black bean hypoglycemic peptides Se-MetIP and TLTSe-MetLR, using the same method as step 3. Figure 3As shown in the results, the selenium-rich black bean hypoglycemic peptides Se-MetIP and TLTSe-MetLR could inhibit the activity of α-amylase in the concentration range of 0.1 mg / mL to 4 mg / mL, and the inhibition rate tended to be flat at higher concentrations. 50 They are 0.54 mg / mL and 0.47 mg / mL respectively.

[0032] At a concentration of 4 mg / mL, the selenium-rich black bean hypoglycemic peptide Se-MetIP has the strongest inhibitory effect on α-amylase, with an inhibition rate of 70.58±2.21%; the selenium-rich black bean hypoglycemic peptide TLTSe-MetLR has the strongest inhibitory effect on α-amylase, with an inhibition rate of 64.98±0.62%. The molecular weight of the two synthetic peptides that have an inhibitory effect on α-amylase is less than 1000, and they are small molecule polypeptides that are easily absorbed and utilized by the intestines and can play a better role.

[0033] Step 7: Circular dichroism spectroscopy is used to measure the changes in the secondary structure content of the selenium-rich black bean hypoglycemic peptides Se-MetIP and TLTSe-MetLR after binding with α-amylase. The specific steps are as follows: 1) Prepare sample solutions of selenium-enriched black bean hypoglycemic peptides Se-MetIP and TLTSe-MetLR respectively; 2) Mix 0.5 mL of sample solution with 0.5 mL of α-amylase solution and react at 37°C for 20 min.

[0034] 3) PBS was used instead of selenium-enriched black bean glucose-lowering peptide as the control group.

[0035] 4) Measurement parameters: path length is 0.1cm, step length is 1nm, wavelength is 190~260nm, the test results are as follows Figure 4 As shown: The content changes of each secondary structure were obtained by analyzing the spectrum with CDNN software (Table 2).

[0036] Table 2 Effects of selenium-enriched black bean glucose-lowering peptide on the secondary structure of α-amylase

[0037] Depend on Figure 4It can be seen that α-amylase has a negative peak at a wavelength of 222 nm. With the addition of glucose-lowering peptides, the intensity of the negative peak decreased significantly, indicating that the secondary structure of α-amylase has changed. In the table, the addition of glucose-lowering peptides reduced the proportion of β-turns and irregular curls and increased the proportion of β-folds. It shows that glucose-lowering peptides induce changes in the secondary structure of α-amylase, change the spatial conformation of the enzyme, and thus affect the binding of the substrate to the active site of the enzyme. The secondary structure is stabilized by hydrogen bonds between the C=O and NH groups on the skeleton, and hydrogen bonds are the main force to maintain the secondary structure. The selenium-rich black bean glucose-lowering peptide and α-amylase are also mainly bound by hydrogen bonds, which indicates that the change in the secondary structure of α-amylase is related to the formation of hydrogen bonds between glucose-lowering peptides and α-amylase.

[0038] Step 8. Determination of the thermodynamic properties of the interaction between selenium-enriched black bean glucose-lowering peptide and α-amylase by isothermal titration calorimetry 1) Dissolve the glucose-lowering peptide Se-MetIP / TLTSe-MetLR and α-amylase solution in PBS buffer.

[0039] 2) At 25°C and 350 r / min, titrate 300 μL of 0.4uM α-amylase with about 50 μL of 0.01mM glucose-lowering peptide solution.

[0040] 3) Titrate the glucose-lowering peptide solution into the α-amylase solution 20 times at 120 s intervals, 2.5 μL each time.

[0041] 4) Set up a blank experimental group by titrating the glucose-lowering peptide solution into PBS buffer for measurement.

[0042] 5) The isothermal titration calorimeter records the change in heat over time, and the Microcal Origin 7.0 software is used to calculate the thermodynamic parameters related to the interaction between the glucose-lowering peptide and α-amylase. The binding constant (Ka), enthalpy change (ΔH), entropy change (ΔS), Gibbs free energy change (ΔG) and number of binding sites (n) are determined by the "One set of sites" mode; Figure 5 The heat flow changes over time when Se-MetIP and TLTSe-MetLR bind to α-amylase are shown. In Table 3, △G and -T*△S are both positive values, and △H is negative, indicating that the reaction between Se-MetIP and α-amylase is an exothermic process. With the continued addition of Se-MetIP, the binding of α-amylase and Se-MetIP tends to saturation, and the heat release gradually decreases. The binding ratio (N) of α-amylase and Se-MetIP obtained by fitting is 1.466, and the corresponding dissociation constant (Kd) is 5.923*10 -6M. The △H value is negative, indicating that the binding reaction is driven by enthalpy and is mainly bound by non-covalent interactions such as hydrogen bonds and van der Waals forces. Therefore, it is speculated that hydrogen bonds and van der Waals forces are the main driving forces for the binding of Se-MetIP to α-amylase.

[0043] △G, △H and -T*△S are all negative values, indicating that the reaction between TLTSe-MetLR and α-amylase is a spontaneous exothermic process. With the continued addition of TLTSe-MetLR, the binding of α-amylase and TLTMLR tends to be saturated, and the exothermic heat gradually decreases. The binding ratio (N) of α-amylase and TLTSe-MetLR obtained by fitting is 2.666, and the corresponding dissociation constant (Kd) is 1.137*10 -6 The negative -T*△S value indicates that the binding reaction is driven by entropy and is mainly bound by non-covalent interactions such as hydrogen bonds and van der Waals forces. Therefore, it is speculated that hydrogen bonds and van der Waals forces are the main driving forces for the binding of TLTSe-MetLR to α-amylase.

[0044] Table 3 Thermodynamic parameters related to the interaction between glucose-lowering peptides and α-amylase

[0045] Step 9: Analysis of the potential binding conformation between selenium-enriched black bean glucose-lowering peptide and α-amylase by molecular docking According to the peptide sequence information, PEP-FOLD3 was used to predict the peptide structure, and the structure was named according to the peptide sequence. Then the MOPAC program was used to optimize the peptide structure and calculate the PM3 atomic charge for subsequent molecular docking. AutoDock Tools 1.5.6 was used to process the ligand structure and generate a pdbqt file for docking. The AutoDock 4.2.6 software package was used to perform global molecular docking between the peptide and the protein. The center coordinates of the docking box were set to the center of the protein to cover the entire protein structure. The number of grid points in each direction of XYZ was set to 100×100×100, the number of dockings was set to 50, and the rest of the parameters used the default values. Figure 6-1 The three-dimensional structure of α-amylase (protein 5U3A). Figure 6-2 It is the structure of selenium-enriched black bean glucose-lowering peptide. The binding site of selenium-enriched black bean glucose-lowering peptide on protein has not been reported in the literature, so the present invention uses a molecular docking method to perform global molecular docking between the polypeptide and the protein. Table 4 gives the molecular docking energy scoring results of the polypeptide and the protein, showing that the binding energy between protein 5U3A and Se-MetIP and TLTSe-MetLR is -5.399 kcal / mol and -6.401 kcal / mol, and the strong binding energy results indicate that the polypeptide and the protein can be stably bound.

[0046] Table 4 Scoring results of docking of selenium-enriched black bean glucose-lowering peptide molecules with α-amylase

[0047] The results of the docking of 5U3A protein and Se-MetIP are as follows Figure 6-3 As shown. The peptide can stably bind to the cavity composed of Gln347, Phe348, Trp316, Asp317, Thr314 and Arg346 amino acids in the protein. Further analysis of the interaction between the two showed that hydrophobic and hydrogen bonding interactions were formed between the peptide and the amino acids around the pocket to promote the stable binding of the peptide and the protein. Specifically, two groups of hydrogen bonds were formed between the peptide and the Arg346 amino acids around the protein; at the same time, the peptide also formed a hydrophobic interaction with the five amino acids around the protein pocket, further enhancing the affinity between the peptide and the protein.

[0048] The results of the molecular docking between 5U3A protein and TLTSe-MetLR are shown in Figure 6-4 As shown. The peptide can stably bind to the cavity composed of Gly308, Ser311, Ala310, Gly304, Thr314, Ile312, Leu313, Arg303, Phe348, Gln302, Asp297, Phe256, Asn301, Val296, Ala307, Ile235, Glu233, Glu309 and Arg346 amino acids in the protein. Further analysis of the interaction between the two showed that hydrophobic and hydrogen bonding interactions were formed between the peptide and the amino acids around the pocket to promote the stable binding of the peptide to the protein. Specifically, four groups of hydrogen bonds were formed between the peptide and the Glu233, Glu309 and Arg346 amino acids around the protein; at the same time, the peptide also formed a hydrophobic interaction with the 16 amino acids around the protein pocket, further enhancing the affinity between the peptide and the protein.

[0049] Step 10: Detect the effect of selenium-enriched black bean glucose-lowering peptide on the viability of HepG2 cells. The specific steps are as follows: 1) 2×10 5 The cells were inoculated in 96-well plates at a density of cells / mL, with 200 μL per well.

[0050] 2) Set up a blank group and a selenium-enriched black bean glucose-lowering peptide group.

[0051] 3) After the cells adhered to the wall, discard the old culture medium and add serum-free culture medium to starve the cells for 10 hours. − 6mol / L insulin serum-free medium, 10 μL per well. The blank group is a medium group without cells; the selenium-enriched black bean glucose-lowering peptide groups are added with Se-MetIP and TLTSe-MetLR, respectively.

[0052] 4) After incubating the 96-well plate in the incubator for an appropriate period of time, add 10 μL of CCK-8 solution to each well of the plate and incubate for 4 hours. Gently shake and measure the absorbance at 450 nm to investigate the effect of selenium-enriched black bean hypoglycemic peptide on cell proliferation.

[0053] from Figure 7 It can be seen that the two selenium-rich black bean glucose-lowering peptides did not affect the proliferation of HepG2 cells. The cell survival rate of the Se-MetIP group was 95.52%, and the cell survival rate of the TLTSe-MetLR group was 98.55%, indicating that the two selenium-rich black bean glucose-lowering peptides had basically no cytotoxicity.

[0054] Step 11: Detect the effect of selenium-enriched black bean glucose-lowering peptide on glucose metabolism in IR-HepG2 cell model. The specific steps are as follows: 1) 2×10 5 The cells were seeded in 96-well plates at a density of 1.5 cells / mL.

[0055] 2) Set up blank group, model group, positive control group, and selenium-enriched black bean glucose-lowering peptide group, with 6 replicate wells in each group. All wells except the blank group were added with 10 −6 mol / L insulin in serum-free medium for 24h. The blank group and model group were added with serum-free medium, the positive control group was added with serum-free medium containing acarbose, and the selenium-enriched black bean glucose-lowering peptide group was added with serum-free medium containing Se-MetIP and TLTSe-MetLR, respectively. The kit was used to measure the glucose and glycogen content, and the effect of selenium-enriched black bean glucose-lowering peptide on the glucose and glycogen consumption of IR-HepG2 cells was calculated. The results of cell glucose consumption are shown in Figure 8-1 As shown in the results, acarbose and two selenium-enriched black bean glucose-lowering peptides can promote the absorption of glucose by IR-HepG2 cells. Compared with the model control group, Se-MetIP significantly improved the glucose uptake of IR-HepG2 cells, indicating that the two selenium-enriched black bean glucose-lowering peptides can improve the glucose consumption of IR-HepG2 cells, and Se-MetIP has a better effect. Therefore, selenium-enriched black bean glucose-lowering peptides may show a hypoglycemic effect by increasing glucose consumption.

[0056] The glycogen level in cells was measured to evaluate the effect of selenium-enriched black bean glucose-lowering peptide on the glycolytic function of insulin-resistant HepG2 cells. Figure 8-2The results showed that compared with the normal HepG2 cells in the blank control group, the glycogen content of IR-HepG2 cells was significantly reduced by 31.74% (p < 0.01), indicating that high concentrations of insulin are not conducive to the glycogen synthesis of normal HepG2 cells. Compared with the model control group, the positive control group, TLTSe-MetLR group and Se-MetIP group can increase the glycogen content of IR-HepG2 cells, among which the positive control group and Se-MetIP group significantly increased the glycogen content (p < 0.01), increasing by 29.18% and 28.17%, respectively. It can be seen that selenium-rich hypoglycemic peptides can improve glucose metabolism in IR-HepG2 cells by increasing the glycogen content in IR-HepG2 cells, thereby achieving a hypoglycemic effect.

[0057] In summary, the present invention uses selenium-rich black beans as raw materials and successfully prepares selenium-rich black bean polypeptides with hypoglycemic activity through specific enzymatic hydrolysis and ultrafiltration processes. The molecular weight of these polypeptides is less than 1 kDa, making them more easily absorbed and utilized by the human body, thereby improving the bioavailability and effect of hypoglycemic products.

[0058] Compared with ordinary black beans, selenium-enriched black beans are rich in selenium, so the peptides extracted from them have better effects in inhibiting α-amylase. α-amylase is one of the key enzymes in the source of glucose in the body. By inhibiting its activity, the production and release of glucose can be reduced, thereby effectively controlling blood sugar levels. This characteristic makes selenium-enriched black bean peptides have great potential in the preparation of efficient blood sugar-lowering products.

[0059] In addition, the present invention screened out two new selenium-rich black bean peptides, Se-MetIP and TLTSe-MetLR, from selenium-rich black bean peptides. These two peptides not only have inhibitory activity against α-amylase, but cell experiments further verified that they may play a hypoglycemic effect by increasing glycogen content. This discovery not only enriches the types of hypoglycemic active ingredients, but also provides new ideas and basis for clarifying the potential mechanism of hypoglycemia.

[0060] Furthermore, the selenium-rich black bean polypeptides of the present invention can be used to prepare blood sugar-lowering products, and have broad application prospects and market potential. They can be applied to multiple fields such as food, health products, and medicines, providing more choices and better treatment effects for diabetic patients and people who need to control blood sugar, which is of great significance for promoting the research and development of blood sugar-lowering products.

[0061] Finally, the preparation method of the present invention is simple, easy to implement, and has relatively low cost, which is conducive to the high-value utilization and industrial development of selenium-rich black beans, and helps to promote the development of the agricultural economy.

Claims

1. A selenium-enriched black bean polypeptide, characterized in that: The selenium-enriched black bean is enzymatically hydrolyzed by alkaline protease and then freeze-dried to obtain the product.

2. The selenium-enriched black soybean polypeptide according to claim 1, characterized in that The dosage of the alkaline protease is 1% to 5%.

3. Use of the selenium-enriched black bean polypeptide according to claim 1 or 2 in the preparation of α-amylase inhibitors.

4. The use according to claim 3, characterized in that: The molecular weight of the selenium-rich black bean polypeptide is less than 1 kDa.

5. Use of the selenium-enriched black bean polypeptide according to claim 1 or 2 in the preparation of a blood sugar-lowering product.

6. The use according to claim 5, characterized in that: The molecular weight of the selenium-rich black bean polypeptide is less than 1 kDa.

7. A product with blood sugar lowering function, characterized in that: The active ingredient comprises the selenium-rich black bean polypeptide according to claim 1 or 2, and the concentration of the selenium-rich black bean polypeptide is 2 mg / mL to 10 mg / mL.

8. A selenium-rich black bean hypoglycemic peptide, characterized in that: The selenium-rich black bean blood sugar lowering peptide is screened from the selenium-rich black bean polypeptide according to claim 1 or 2, including the selenium-rich black bean blood sugar lowering peptide Se-MetIP and / or the selenium-rich black bean blood sugar lowering peptide TLTSe-MetLR.

9. Use of the selenium-rich black bean hypoglycemic peptide according to claim 8 in the preparation of products with hypoglycemic function or α-amylase inhibitors.

10. A product with blood sugar lowering function, characterized in that: The active ingredient comprises the selenium-rich black bean blood sugar lowering peptide as claimed in claim 8, and the concentration of the selenium-rich black bean blood sugar lowering peptide is 0.1 mg / mL to 4 mg / mL.

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