A kind of inhibition alpha-glucosidase bird's nest peptide and its preparation method and application

Abstract

The application discloses a kind of inhibiting alpha-glucosidase bird's nest peptide and its preparation method and application, belong to functional polypeptide technical field.The amino acid sequence of inhibiting alpha-glucosidase bird's nest peptide provided in the application is WLR.The molecular weight of the bird's nest peptide of the application is 473.275 Da, it enters the active center of alpha-glucosidase, occupies hydrophobic pocket, and produces bond with surrounding amino acid residues, is combined more closely, with the advantages of controllable blood glucose level and safe non-toxic side effect.The application further provides a kind of inhibiting alpha-glucosidase bird's nest peptide powder, the bird's nest peptide powder includes inhibiting alpha-glucosidase bird's nest peptide described above, the bird's nest peptide powder has the effect of inhibiting alpha-glucosidase, can play the role of reducing blood sugar, can be applied to the preparation of blood sugar reducing product.

Description

Technical Field

[0001] This invention belongs to the field of functional polypeptide technology, and specifically relates to a bird's nest peptide that inhibits α-glucosidase, its preparation method, and its application. Background Technology

[0002] Diabetes, a global chronic metabolic disease, is experiencing a significant increase in incidence. In recent years, the number of people with diabetes worldwide has increased dramatically, placing a heavy economic burden on society and families. Diabetes is mainly divided into type 1 and type 2 diabetes, with type 2 accounting for over 90% of cases. Prolonged hyperglycemia can lead to a series of serious complications, such as cardiovascular disease, neuropathy, nephropathy, and retinopathy. These complications not only severely impact a patient's quality of life but can even be life-threatening.

[0003] Alpha-glucosidase is a carbohydrate hydrolase found in the brush border of the small intestinal mucosa. It breaks down carbohydrates such as polysaccharides and oligosaccharides in food into monosaccharides such as glucose, which can then be absorbed and utilized by the body. Under normal physiological conditions, alpha-glucosidase plays an important role in maintaining the body's energy supply. However, in diabetic patients, the activity of alpha-glucosidase is often too high, leading to accelerated digestion and absorption of carbohydrates, a rapid rise in postprandial blood glucose, and exacerbated blood glucose fluctuations. Therefore, inhibiting the activity of alpha-glucosidase, delaying the digestion and absorption of carbohydrates, and reducing postprandial blood glucose peaks has become one of the important strategies for treating type 2 diabetes.

[0004] Currently, commonly used alpha-glucosidase inhibitors in clinical practice include acarbose and voglibose. These drugs are effective in lowering postprandial blood glucose, but they also have some significant limitations. Firstly, these chemically synthesized drugs may cause a range of gastrointestinal adverse reactions, such as bloating, diarrhea, and abdominal pain, affecting patient adherence. Secondly, long-term use of chemically synthesized drugs may also cause damage to organs such as the liver and kidneys, increasing potential health risks. Furthermore, with the increasing number of diabetic patients, the demand for novel, safe, and effective alpha-glucosidase inhibitors is becoming increasingly urgent.

[0005] Bird's nest is a traditional and precious tonic, rich in nutrients and offering various health benefits. It contains various proteins, amino acids, minerals, and other nutrients, and has long been considered to have effects such as nourishing yin and moisturizing dryness, replenishing qi and strengthening the spleen, and beautifying the skin. In recent years, with the deepening of research on bird's nest, it has been discovered that the proteins in bird's nest can produce various bioactive peptides after enzymatic hydrolysis. These bioactive peptides possess multiple physiological functions, including antioxidant, immunomodulatory, and antibacterial properties. However, whether bird's nest peptides can inhibit α-glucosidase activity, and their potential application in lowering blood sugar, has not yet been extensively reported in research. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides an α-glucosidase-inhibiting bird's nest peptide, its preparation method, and its application. The bird's nest peptide of this invention exhibits a strong inhibitory effect on α-glucosidase, possesses the advantages of controlling blood sugar levels, and is safe and free of toxic side effects. It can be used as a product to lower blood sugar, thereby expanding the added value of bird's nest.

[0007] The technical solution of the present invention is as follows: One objective of this invention is to provide an α-glucosidase inhibitory bird's nest peptide, wherein the amino acid sequence of the α-glucosidase inhibitory bird's nest peptide is SEQ ID NO:1.

[0008] A second objective of this invention is to provide a bird's nest peptide powder that inhibits α-glucosidase, wherein the bird's nest peptide powder comprises the bird's nest peptide that inhibits α-glucosidase as described in claim 1.

[0009] The third objective of this invention is to provide a method for preparing bird's nest peptide powder that inhibits α-glucosidase, wherein the bird's nest peptide powder is obtained by ultrafiltration chromatography from the enzymatic hydrolysate of dried bird's nest powder.

[0010] Furthermore, the preparation method includes the following steps: S1: Weigh out the dried bird's nest powder, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kw and the time is 25s. S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 7.5-8.5, add trypsin, and enzymatically hydrolyze at 40-50℃ for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a trypsin hydrolysate is obtained; wherein, the amount of trypsin added is 1-2% of the mass of the bird's nest. S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 1.5-2.5, add pepsin, and enzymatically hydrolyze at 35-40℃ for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, the pepsin hydrolysate is obtained. The amount of pepsin added is 1-2% of the weight of the bird's nest. S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and after ultrafiltration and chromatography, obtain the bird's nest peptide powder that inhibits α-glucosidase.

[0011] Furthermore, in step S2, the trypsin has an enzyme activity ≥2500 U / mg.

[0012] Furthermore, in step S3, the pepsin enzyme activity is ≥3000 U / mg.

[0013] Further, in step S4, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 3 kDa to collect enzymatic hydrolysis products with a molecular weight less than 3 kDa.

[0014] Further, in step S4, the chromatography process is as follows: the pepsin hydrolysate after ultrafiltration is subjected to column chromatography using a Superdex™ peptide 10 / 300 GL gel chromatography column equipped with an AKTA system, and a total of 4 elution peaks are obtained. The eluents corresponding to the 4 elution peaks are collected and freeze-dried to obtain 4 peptides. Then, using α-glucosidase inhibitory activity as the evaluation index, the bird's nest peptide that inhibits α-glucosidase is screened from the 4 peptides through in vitro α-glucosidase inhibition experiments. The column chromatography conditions were as follows: sample concentration of 20 mg / mL, sample volume of 1 mL, flow rate of 300 μL / min, collection of one tube every 200 μL, detection at 220 nm and plotting of absorbance curves.

[0015] Furthermore, the in vitro α-glucosidase inhibition experiment includes the following steps: four peptides are mixed with water to prepare four peptide solutions, 20 μL of each of the four peptide solutions are mixed with 20 μL of α-glucosidase, and then added to 96-well plates and shaken to mix. After incubation at room temperature for 20 min, 20 μL of PNPG is added and mixed thoroughly. The mixture is then incubated at 37°C in the dark for 20 min. Finally, 100 μL of 1M sodium carbonate solution is added to terminate the reaction and the absorbance at 410 nm is immediately detected.

[0016] The fourth objective of this invention is to provide an application of the above-mentioned α-glucosidase-inhibiting bird's nest peptide, the above-mentioned α-glucosidase-inhibiting bird's nest peptide powder, or any of the above-mentioned preparation methods of bird's nest peptide powder in the preparation of blood sugar-lowering products.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides an α-glucosidase inhibitory bird's nest peptide with a molecular weight of 473.275 Da. It enters the active site of α-glucosidase, occupies the hydrophobic pocket, and forms a bond with the surrounding amino acid residues. The binding is relatively tight, and it has the advantages of controlling blood sugar levels and being safe and free of toxic side effects.

[0018] 2. The bird's nest peptide powder provided by this invention has the effect of inhibiting α-glucosidase and can be used as a product to lower blood sugar, which can broaden the added value of bird's nest.

[0019] 3. This invention provides a method for preparing bird's nest peptide powder that inhibits α-glucosidase. The method involves stepwise enzymatic hydrolysis of bird's nest fragments, followed by ultrafiltration and chromatography to obtain bird's nest peptide powder that inhibits α-glucosidase. This method maximizes the utilization of the nutritional components of bird's nest, broadens the development avenues for hypoglycemic products, and enhances the economic value and market position of bird's nest. Attached Figure Description

[0020] Figure 1 This is a column chromatography elution diagram of the enzymatic hydrolysate from Example 1 of this invention; Figure 2 This invention relates to the α-glucosidase inhibitory activity of four polypeptides labeled 1-1, 1-2, 1-3, and 1-4 separated by chromatography in Example 1 (the types and labels of polypeptides separated in Example 2 are the same). Figure 3 This is a secondary mass spectrum of the bird's nest polypeptide from Example 1 of this invention; Figure 4 This invention provides a diagram showing the conformation of the bird's nest polypeptide in the α-glucosidase pocket, its binding mode at the active site, and its interaction planar diagram in Example 1. Figure 5 In this invention, the α-glucosidase inhibitory activities of EBNP-1 in Example 2, EBNP-2 in Comparative Example 1, and EBNP-3 in Comparative Example 2 are shown in ad, indicating significant differences between groups. Figure 6 In this invention, EBNP-1 prepared in Example 2 is shown to be cytotoxic to LO2 cells. Detailed Implementation

[0021] The following describes a preferred embodiment, with reference to the appendix. Figure 1-6To further illustrate the present invention, the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values; for numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein; the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified; the experimental methods in the following embodiments are conventional methods unless otherwise specified.

[0022] This invention provides a bird's nest peptide that inhibits α-glucosidase, wherein the amino acid sequence of the bird's nest peptide that inhibits α-glucosidase is WLR (SEQ ID NO:1).

[0023] This invention obtains bird's nest peptide powder by ultrafiltration chromatography of the enzymatic hydrolysate of dried bird's nest powder.

[0024] The α-glucosidase-inhibiting bird's nest peptide provided by this invention can also be used to prepare blood sugar-lowering products.

[0025] The following specific embodiments further illustrate the α-glucosidase-inhibiting bird's nest peptide of the present invention and its applications: The experimental indicators and methods involved in the following embodiments are as follows: (1) Gel column chromatography The gel chromatography column used was a Superdex™ peptide 10 / 300 GL gel chromatography column. The chromatography method was as follows: the sample concentration was 20 mg / mL, the sample volume was 1 mL, the flow rate was 300 μL / min, and one tube was collected every 200 μL. The absorbance curves of each chromatographic component were detected under a 220 nm wavelength UV detector and plotted.

[0026] (2) In vitro α-glucosidase inhibition experiment 20 μL of peptide solution and 20 μL of α-glucosidase were added sequentially to a 96-well plate and vortexed to mix. After incubation at room temperature for 20 min, 20 μL of PNPG was added and thoroughly mixed. The plate was then incubated at 37°C in the dark for 20 min. Finally, 100 μL of 1M sodium carbonate solution was added to terminate the reaction, and the absorbance at 410 nm was immediately measured. PBS buffer was used instead of samples for the blank control group, and PBS buffer was used instead of samples for the complete reaction group of α-glucosidase and p-nitrophenylglucose. Acarbose was used instead of samples for the positive control.

[0027] α-glucosidase inhibition rate (%) = [1 - (D2–D1) / D3] × 100% In the formula: D1 is the sample control group; D2 is the sample reaction group; D3 is the complete reaction group.

[0028] Example 1 This embodiment provides a bird's nest peptide that inhibits α-glucosidase. The preparation process of the bird's nest peptide that inhibits α-glucosidase includes the following steps: 1) Preparation of bird's nest peptide powder: S1: Weigh out the dried bird's nest powder, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kw and the time is 25s. S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 8.5, add trypsin, enzymatically hydrolyze at 50°C for 4 hours, and then inactivate the enzyme with boiling water for 10 minutes to obtain trypsin hydrolysate; wherein, the amount of trypsin added is 1% of the mass of bird's nest; and the enzyme activity of the trypsin is ≥2500 U / mg. S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 2.0, add pepsin, and perform enzymatic hydrolysis at 37°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a pepsin hydrolysate is obtained. The amount of pepsin added is 1% of the weight of the bird's nest. The enzyme activity of the pepsin is ≥3000 U / mg. S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and after ultrafiltration and chromatography, obtain the bird's nest peptide powder that inhibits α-glucosidase.

[0029] In this embodiment, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, enzymatic hydrolysis products with a molecular weight less than 3 kDa are collected.

[0030] The chromatography process is as follows: The ultrafiltration-treated pepsin hydrolysate is subjected to column chromatography using a Superdex™ peptide 10 / 300 GL gel chromatography column equipped with an AKTA system. The resulting absorbance curve is shown in the figure below. Figure 1 As shown, four elution peaks were obtained. The eluents corresponding to the four elution peaks were collected, freeze-dried, and four peptides were obtained, labeled as 1-1, 1-2, 1-3, and 1-4, respectively. Then, α-glucosidase inhibitory activity was used as the evaluation index, as shown in the figure. Figure 2 As shown, the four peptides were verified by in vitro α-glucosidase inhibition experiments, and peptides 1-4 were found to be the components with the highest α-glucosidase inhibitory activity. The column chromatography conditions were as follows: sample concentration of 20 mg / mL, sample volume of 1 mL, flow rate of 300 μL / min, collection of one tube every 200 μL, detection at 220 nm and plotting of absorbance curves.

[0031] 2) Amino acid sequence identification The selected peptides 1-4 were measured by LC-MS / MS, and the results were analyzed using PEAKS Studio11 mass spectrometry software.

[0032] In this embodiment, the LC-MS / MS measurement conditions are as follows: In the liquid chromatography method: the column was a NanoViper C18 1.9 μm, 100A, with phase A being 0.1% FA and phase B being 0.1% FA and 80% ACN. The flow rate was 50 nL / min, with a chromatographic gradient of 70 min. The specific elution gradient was as follows: 0-1 min, phase B increased uniformly from 8% to 12%; 1-9 min, phase B increased uniformly from 12% to 15%; 9-31 min, phase B increased uniformly from 15% to 25%; 31-38 min, phase B increased uniformly from 25% to 36%; 38-45 min, phase B increased uniformly from 36% to 60%; 45-53 min, phase B increased uniformly from 60% to 95%; 53-60 min, the mobile phase remained unchanged; 60-70 min, phase B decreased uniformly from 95% to 8%.

[0033] In the mass spectrometry method: First-order mass spectrometry parameters: Resolution: 120000; AGC target: Custorm; Maximum IT: Custorm; Scan range: 100 to 1000 m / z. Second-order mass spectrometry parameters: Resolution: 15000; AGC target: Custorm; Maximum IT: Custorm; Cycle time: 3 s; NCE / stepped NCE: 30.

[0034] Search criteria: The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: Fixed modifications: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (Peptide N-term); Enzyme: Non-specific; Database: Apodiformes; Peptide Mass Tolerance (PMT): 20 ppm; Fragment Mass Tolerance (FMT): 0.02 Da.

[0035] The secondary mass spectrum of the bird's nest polypeptide in this embodiment is as follows: Figure 3 As shown.

[0036] 3) Molecular docking screening The activity of several peptides obtained from the PEAKS Studio 11 mass spectrometry analysis software was evaluated using the PeptideRanker tool, with a score greater than 0.9 (indicating a high probability of biological activity). Furthermore, the potential toxicity of the peptides was predicted using the ToxinPred tool to exclude peptides with potential application risks; all results showed no toxicity. Peptides with a score greater than 0.5 were screened using the CPPpred tool (indicating good cell penetration). New peptides were then screened using the Biopep database.

[0037] Based on molecular docking, the software Autodock (version 1.5.7) was used to screen new peptides obtained from mass spectrometry identification and simulation screening for bioactivity, identifying peptide sequences with strong protein binding ability. The spatial structure of the peptides was drawn using ChemDraw 22 software, and energy minimization was performed. Protonation states and hydrogen orientations were optimized in Autodock, and the results were saved as pdbqt files as ligand files.

[0038] The 3D structure of Alpha-glucosidase (PDB ID: 3WY1) was obtained from the PDB database. Repeating sequences and water molecules were removed, and polar hydrogen atoms were added. A gridbox was set within the predicted active pocket of Alpha-glucosidase, with active sites at X: 1.098, Y: -4.174, and Z: -7.923; the grid size was 21.75 Å × 25.5 Å × 18.75 Å.

[0039] Figure 4 The conformation of the bird's nest polypeptide in the α-glucosidase pocket, its binding mode at the active site, and its interaction planar diagram in this embodiment are shown below. Figure 4 As shown, WLR has the strongest binding energy to Alpha-glucosidase, at -8.1 kcal / mol. The active peptide enters the active site of α-glucosidase, occupies the hydrophobic pocket, and forms bonds with the surrounding amino acid residues, resulting in a relatively tight binding and potential hypoglycemic activity.

[0040] Example 2 This embodiment provides a bird's nest peptide powder that inhibits α-glucosidase, wherein the bird's nest peptide powder contains the bird's nest peptide that inhibits α-glucosidase as described in Example 1.

[0041] The preparation method of the bird's nest peptide powder that inhibits α-glucosidase includes the following steps: S1: Weigh out the dried bird's nest powder, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kw and the time is 25s. S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 8.5, add trypsin, enzymatically hydrolyze at 50°C for 4 hours, and then inactivate the enzyme with boiling water for 10 minutes to obtain trypsin hydrolysate; wherein, the amount of trypsin added is 1% of the mass of bird's nest; and the enzyme activity of the trypsin is ≥2500 U / mg. S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 2.0, add pepsin, and perform enzymatic hydrolysis at 37°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a pepsin hydrolysate is obtained. The amount of pepsin added is 1% of the weight of the bird's nest. The enzyme activity of the pepsin is ≥3000 U / mg. S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and after ultrafiltration and chromatography, obtain the bird's nest peptide powder that inhibits α-glucosidase.

[0042] In this embodiment, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, enzymatic hydrolysis products with a molecular weight less than 3 kDa are collected.

[0043] The chromatography process is as follows: The ultrafiltration-treated pepsin hydrolysate is subjected to column chromatography using a Superdex™ peptide 10 / 300 GL gel chromatography column equipped with an AKTA system. The resulting absorbance curve is shown in the figure below. Figure 1 As shown, four elution peaks were obtained. The eluents corresponding to the four elution peaks were collected, freeze-dried, and four peptides were obtained, labeled as 1-1, 1-2, 1-3, and 1-4, respectively. Then, α-glucosidase inhibitory activity was used as the evaluation index, as shown in the figure. Figure 2 As shown, the four peptides were verified by in vitro α-glucosidase inhibition experiments, and peptides 1-4 were found to be the components with the highest α-glucosidase inhibitory activity. The column chromatography conditions were as follows: sample concentration of 20 mg / mL, sample volume of 1 mL, flow rate of 300 μL / min, collection of one tube every 200 μL, detection at 220 nm and plotting of absorbance curves.

[0044] Example 3 This embodiment provides a bird's nest peptide powder that inhibits α-glucosidase, wherein the bird's nest peptide powder contains the bird's nest peptide that inhibits α-glucosidase as described in Example 1.

[0045] The preparation method of the bird's nest peptide powder that inhibits α-glucosidase includes the following steps: S1: Weigh out the dried bird's nest powder, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kw and the time is 25s. S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 7.5, add trypsin, and enzymatically hydrolyze at 37°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a trypsin hydrolysate is obtained; wherein, the amount of trypsin added is 1.5% of the mass of the bird's nest. S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 1.5, add pepsin, and enzymatically hydrolyze at 35°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a pepsin hydrolysate is obtained. The amount of pepsin added is 1.5% of the weight of the bird's nest. S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and after ultrafiltration and chromatography, obtain the bird's nest peptide powder that inhibits α-glucosidase.

[0046] In this embodiment, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, enzymatic hydrolysis products with a molecular weight less than 3 kDa are collected.

[0047] The absorbance of the components obtained by gel chromatography under a 220 nm ultraviolet wavelength detector and the results of the in vitro α-glucosidase inhibition experiment in this embodiment are similar to those in Example 2.

[0048] Example 4 This embodiment provides a bird's nest peptide powder that inhibits α-glucosidase, wherein the bird's nest peptide powder contains the bird's nest peptide that inhibits α-glucosidase as described in Example 1.

[0049] The preparation method of the bird's nest peptide powder that inhibits α-glucosidase includes the following steps: S1: Weigh out the dried bird's nest powder, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kw and the time is 25s. S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 8, add trypsin, and enzymatically hydrolyze it at 40°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a trypsin hydrolysate is obtained; wherein, the amount of trypsin added is 2% of the mass of the bird's nest. S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 2.5, add pepsin, and enzymatically hydrolyze at 40°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a pepsin hydrolysate is obtained. The amount of pepsin added is 2% of the weight of the bird's nest. S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and after ultrafiltration and chromatography, obtain the bird's nest peptide powder that inhibits α-glucosidase.

[0050] In this embodiment, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, enzymatic hydrolysis products with a molecular weight less than 3 kDa are collected.

[0051] The absorbance of the components obtained by gel chromatography under a 220 nm ultraviolet wavelength detector and the results of the in vitro α-glucosidase inhibition experiment in this embodiment are similar to those in Example 2.

[0052] Comparative Example 1 The difference from Example 2 is that: In this comparative example, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 3 kDa and an ultrafiltration tube with a molecular weight cutoff of 10 kDa, the enzymatic hydrolysis products with a molecular weight between 3 kDa and 10 kDa are collected.

[0053] Comparative Example 2 The difference from Example 2 is that: In this comparative example, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, enzymatic hydrolysis products with a molecular weight greater than 10 kDa are collected.

[0054] Implementation effect evaluation The following specific experiments on the bird's nest peptide powders prepared in Examples 2-4 and Comparative Examples 1-2 further illustrate the excellent effects achieved by the present invention: 1) In vitro α-glucosidase inhibition experiment: The bird's nest peptide powder that inhibits α-glucosidase from Example 2 was selected for an in vitro α-glucosidase inhibition experiment and compared with the bird's nest peptide powders of Comparative Examples 1-2. The three samples were designated as EBNP-1, EBNP-2, and EBNP-3, respectively.

[0055] The results are as follows Figure 5 As shown, from Figure 5 As can be seen, the EBNP-1 prepared in Example 2 has a good inhibitory effect on α-glucosidase.

[0056] 2) Cytotoxicity assay: Cytotoxicity assays were performed on the EBNP-1 cells prepared in Example 2. L02 cells in logarithmic growth phase were used to prepare a homogeneous single-cell suspension. After counting, the suspension was diluted with culture medium to 10,000 cells / 100 μL, and 100 μL was seeded per well in a 96-well plate. After culturing for 12-16 h, the old culture medium was discarded, and different concentrations of peptide-containing culture medium (culture medium: peptide solution = 99:1) were added, and the cells were cultured for another 24 h. Finally, under light-protected conditions, 100 μL of fresh culture medium and 15 μL of 5 mg / mL thiazolyl blue solution were added to each well, and the cells were incubated for 4 h. The supernatant was then discarded, and 150 μL of DMSO was added to each well, with shaking for 10 min in the dark. The absorbance was measured at 490 nm. Six parallel wells were set up for each sample group, and a PBS control group was included.

[0057] The formula for calculating cell viability is as follows: Cell viability (%) = [(A1–A0) / (A2–A0)] × 100% In the formula: A0 is the blank group; A1 is the sample group; A2 is the control group.

[0058] The results are as follows Figure 6 As shown, from Figure 6 It can be seen that the peptide has a growth-promoting effect on LO2 cells at concentrations of 0.1-10 μg / mL, and is non-toxic to cells at a concentration of 100 μg / mL, with cell viability between 95-110%.

[0059] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing bird's nest peptide powder that inhibits α-glucosidase, characterized in that, The bird's nest peptide powder contains an α-glucosidase inhibitor bird's nest peptide with the amino acid sequence SEQ ID NO:

1. The bird's nest peptide powder is obtained by ultrafiltration chromatography from the enzymatic hydrolysate of dried bird's nest fragments. The preparation method includes the following steps: S1: Weigh out the dried bird's nest powder, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kw and the time is 25s. S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 7.5-8.5, add trypsin, and enzymatically hydrolyze at 40-50℃ for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a trypsin hydrolysate is obtained; wherein, the amount of trypsin added is 1-2% of the mass of the bird's nest. S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 1.5-2.5, add pepsin, and enzymatically hydrolyze at 35-40℃ for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, the pepsin hydrolysate is obtained. The amount of pepsin added is 1-2% of the weight of the bird's nest. S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and after ultrafiltration and chromatography, obtain the bird's nest peptide powder that inhibits α-glucosidase.

2. The method for preparing bird's nest peptide powder that inhibits α-glucosidase according to claim 1, characterized in that, In step S2, the trypsin has an enzyme activity ≥2500 U / mg.

3. The method for preparing bird's nest peptide powder that inhibits α-glucosidase according to claim 1, characterized in that, In step S3, the enzyme activity of pepsin is ≥3000 U / mg.

4. The method for preparing bird's nest peptide powder that inhibits α-glucosidase according to claim 1, characterized in that, In step S4, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 3 kDa to collect enzymatic hydrolysis products with a molecular weight less than 3 kDa.

5. A method for preparing bird's nest peptide powder that inhibits α-glucosidase according to claim 1, characterized in that, In step S4, the chromatography process is as follows: the pepsin hydrolysate after ultrafiltration is subjected to column chromatography using a Superdex™ peptide 10 / 300GL gel chromatography column equipped with an AKTA system, and a total of 4 elution peaks are obtained. The eluents corresponding to the 4 elution peaks are collected and freeze-dried to obtain 4 peptides. Then, using α-glucosidase inhibitory activity as the evaluation index, the bird's nest peptide that inhibits α-glucosidase is screened from the 4 peptides through in vitro α-glucosidase inhibition experiments. The column chromatography conditions were as follows: sample concentration of 20 mg / mL, sample volume of 1 mL, flow rate of 300 μL / min, collection of one tube every 200 μL, detection at 220 nm and plotting of absorbance curves.

6. A method for preparing bird's nest peptide powder that inhibits α-glucosidase according to claim 5, characterized in that, The in vitro α-glucosidase inhibition experiment includes the following steps: four peptides are prepared by mixing them with water to form four peptide solutions. 20 μL of each of the four peptide solutions is mixed with 20 μL of α-glucosidase and added to a 96-well plate. The mixture is shaken and incubated at room temperature for 20 min. Then, 20 μL of PNPG is added and thoroughly mixed. The mixture is incubated at 37°C in the dark for 20 min. Finally, 100 μL of 1M sodium carbonate solution is added to terminate the reaction, and the absorbance at 410 nm is immediately detected.

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