Lumbricus-sourced alpha-glucosidase inhibitory peptide and application thereof
By employing an integrated autolysis-enzymatic hydrolysis-ultrafiltration process and virtual screening technology, α-glucosidase inhibitory peptides RGF and FLP derived from earthworm protein were extracted. This solved the problem of the lack of earthworm-derived peptide sequences, achieved efficient and low-cost α-glucosidase inhibition, and filled the research gap in earthworm hypoglycemic active peptides.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU BAIYUNSHAN WEI YI IND CO LTD
- Filing Date
- 2026-01-22
- Publication Date
- 2026-05-12
AI Technical Summary
In the existing technology, research on α-glucosidase inhibitory peptides derived from earthworms is relatively scarce. Traditional preparation processes are cumbersome and costly, and specific α-glucosidase inhibitory peptide sequences are lacking. Virtual screening technology has not been applied in this field.
An integrated autolysis-enzymatic hydrolysis-ultrafiltration process was used to extract α-glucosidase inhibitory peptides from earthworm protein. Combined with virtual screening technology, the peptide sequences Arg-Gly-Phe (RGF) and Phe-Leu-Pro (FLP) with high inhibitory activity were screened using tools such as LC/MSMS, Peptide Ranker, and AutodockVina. The peptides were then prepared by solid-phase synthesis.
The peptides RGF and FLP, which significantly inhibit α-glucosidase activity, were obtained, with IC50 values of 0.599 mg/mL and 1.344 mg/mL, respectively, which are superior to existing peptides. This study simplifies the preparation process, reduces costs, and enriches the library of food-derived α-glucosidase inhibitory peptides.
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Figure CN122011089A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to bioactive peptides and their applications, specifically to α-glucosidase inhibitory peptides derived from earthworms and their applications. Background Technology
[0002] Type 2 diabetes is a metabolic disease characterized by hyperglycemia caused by insulin resistance, often accompanied by metabolic disorders of carbohydrates, proteins, and fats. Long-term hyperglycemia and metabolic disturbances can further induce systemic organ damage, including damage to the kidneys, cardiovascular system, eyes, nervous system, and skin infections. Therefore, lowering blood glucose levels within a scientifically sound framework plays a positive role in the prevention and treatment of diabetes. Inhibiting alpha-glucosidase activity is considered an effective method for treating type 2 diabetes. Clinically, most commonly used alpha-glucosidase inhibitors are chemically synthesized drugs. While these drugs are highly effective, long-term use can cause adverse reactions and side effects such as liver damage, myocardial infarction, edema, and anemia. Therefore, screening for safe, non-toxic natural active ingredients with alpha-glucosidase inhibitory activity has been a major focus of researchers.
[0003] Food-derived α-glucosidase inhibitory peptides have attracted widespread attention from researchers due to their natural, safe, and highly effective characteristics. For example, the tripeptide GEY (IC) isolated from silkworm cocoon hydrolysate... 50 (2.70 mg / mL), GYG (IC50) 50 (1.50 mg / mL), and the peptide PFP (IC50) obtained from Aspergillus oryzae fermentation products. 50 The concentration was 3.10 mg / mL, and all of them showed good α-glucosidase inhibitory activity (Lee, HJ, Lee, HS, Choi, JW, Ra, KS, Kim, JM, & Suh, HJ (2011). Novel tripeptides with alpha-glucosidase inhibitory activity isolated from silk cocoon hydrolysate. Journal of Agricultural and Food Chemistry, 59 (21), 11522). 11525; Kang, MG, Yi, SH, Lee, JS (2013). Production and characterization of a new α-glucosidase inhibitory peptide from Aspergillusoryzae N159 1, Mycobiology, 41:3, 149 154). However, the traditional preparation process of food-derived peptides has significant limitations: it requires a series of complex steps such as protein extraction, enzymatic hydrolysis, separation and purification of hydrolysates, and characterization and identification of active peptides. The operation is cumbersome, time-consuming, and costly, which limits its practical application and transformation.
[0004] The emergence of virtual screening technology has provided a new approach for the discovery of bioactive peptides. Virtual screening is a method for rapidly screening target bioactive molecules from complex databases by evaluating the interaction between ligands and receptors. This method has been widely used to screen and discover naturally occurring bioactive molecules.
[0005] Earthworms (Eisenia fetidae), a traditional animal-derived medicinal material in my country, have a long history of application. Their protein content is as high as 53.5%–63.1%, and they have been proven to contain various components with anticoagulant, thrombolytic, and immunomodulatory activities. In 2009, my country listed earthworm protein as a new resource food, providing policy support for its application in functional foods and pharmaceuticals. The abundant protein resources of earthworms provide a good material basis for the exploration of bioactive peptides; however, research on α-glucosidase inhibitory peptides from earthworms is still relatively scarce. While there are reports on the preparation of bioactive peptides from animal-derived protein hydrolysates, existing technologies mostly focus on optimizing enzymatic hydrolysis processes and have not clearly identified specific earthworm peptide sequences with α-glucosidase inhibitory activity. Furthermore, no research reports have been found on methods for targeted screening of α-glucosidase inhibitory peptides from earthworms using virtual screening technology. Summary of the Invention
[0006] The purpose of this invention is to address the shortcomings of the prior art by providing an α-glucosidase inhibitory peptide derived from earthworms and its application, thus filling the gap in the specific α-glucosidase inhibitory peptide sequence in earthworms.
[0007] This invention provides an α-glucosidase inhibitory peptide derived from earthworms. The amino acid sequence of the α-glucosidase inhibitory peptide is Arg-Gly-Phe or Phe-Leu-Pro; Arg-Gly-Phe is abbreviated as RGF, and Phe-Leu-Pro is abbreviated as FLP.
[0008] Preferably, the amino acid sequence is obtained from earthworm protein hydrolysate EPH using virtual screening technology.
[0009] More preferably, the earthworm protein hydrolysate EPH is prepared by the following steps:
[0010] (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at low temperature; (2) Autolytic enzymatic hydrolysis: Add water to the earthworm homogenate, stir to carry out pre-enzymatic hydrolysis, and terminate the reaction by boiling water bath after the pre-enzymatic hydrolysis is completed and cool to room temperature; (3) Secondary enzymatic hydrolysis: After the autolytic enzymatic hydrolysis is completed, flavor protease is added to the system, the pH and temperature of the enzymatic hydrolysis system are adjusted, and secondary enzymatic hydrolysis is carried out under stirring conditions; after the enzymatic hydrolysis reaction is completed, the reaction is terminated by boiling water bath and cooled; the supernatant is collected by centrifugation, which is the earthworm protease hydrolysate EPH.
[0011] More preferably, in step (2), the mass ratio of earthworm homogenate to water is 1:1 to 1:4.
[0012] More preferably, in step (3), the pH and temperature of the enzymatic hydrolysis system are adjusted, with the pH value being 7.5-10.5 and the temperature being 45-60℃.
[0013] More preferably, in step (3), the amount of flavor protease added is 3000 u / g-7000 u / g based on protein content, and the enzymatic hydrolysis time is 2-5 hours.
[0014] More preferably, the virtual screening technology includes the following steps: (1) Ultrafiltration fractionation: The earthworm protein hydrolysate was centrifuged with EPH and different molecular weight fractions were retained using an ultrafiltration membrane to verify α-glucosidase activity. The fraction with the best α-glucosidase activity inhibition effect was freeze-dried. (2) Identification of active peptide sequences of earthworm: The peptide sequence of the component with the best inhibitory effect on α-glucosidase activity was identified by nano-liquid chromatography-mass spectrometry (nanoLC-MS / MS); the amino acid sequence of potential active peptides was obtained by database search and analysis; (3) Screening of potential active peptides: After screening peptides with 10lgP>20, the potential active peptide sequences identified were scored using the PeptideRanker online tool (http: / / distilldeep.ucd.ie / PeptideRanker). Peptide sequences with a score>0.9 were selected as candidate active peptides. Potential active peptides were screened based on the amino acid composition characteristics of α-glucosidase inhibitory peptides. (4) Docking analysis: The candidate active peptides were docked with the α-glucosidase protein with PDB ID 3A4J using AutodockVina molecular docking software. The docking score (Vina score) was calculated, and peptide sequences with Vina score < -8.9 were screened. (5) Activity verification: The peptide sequences obtained from the above screening were subjected to in vitro α-glucosidase inhibitory activity verification, and their half-maximal inhibitory concentration (IC50) was determined. 50 ), finally determining IC 50Peptides with low values and strong inhibitory activity are targeted as α-glucosidase inhibitory peptides.
[0015] More preferably, in step (3), the amino acid composition features are: ① C-terminus Arg(R) / Lys(K) / Pro(P); ② N-terminus containing Leu(L) or at least one hydroxyl group or basic side chain; ③ Pro is located at the second to last position from the N-terminus to the C-terminus. More preferably, in step (5), the method for determining the α-glucosidase inhibitory activity is as follows: a certain concentration of polypeptide solution and α-glucosidase solution are added to an enzyme-labeled plate, mixed and incubated, and then PNPG (p-nitrophenyl-α-D-glucopyranoside) solution is added for reaction. Colorless PNPG releases nitrophenol (PNP) after being hydrolyzed by α-glucosidase. PNP is yellow under alkaline conditions. The change in enzyme activity is calculated by measuring the amount of PNP produced at 405 nm, and peptides with α-glucosidase inhibitory activity are screened out.
[0016] Preferably, the α-glucosidase inhibitory peptide derived from earthworms can be artificially synthesized according to the above-mentioned amino acid sequence. The artificial synthesis includes synthesis using solid-phase synthesis methods, wherein the solid-phase synthesis methods include Fmoc-SPPS method, BOC-SPPS method and fragment condensation linking method.
[0017] This invention also provides the application of the above-mentioned earthworm-derived α-glucosidase inhibitory peptide in the preparation of adjuvant hypoglycemic products.
[0018] Preferably, the preparation of blood sugar-lowering products includes using α-glucosidase inhibitory peptides as active ingredients in the preparation of foods, special medical purpose formula foods, or pharmaceuticals that help lower blood sugar.
[0019] More preferably, the drug is a preparation made with an effective amount of earthworm α-glucosidase inhibitory peptide as the active ingredient, plus medically acceptable excipients or auxiliary ingredients.
[0020] Compared with the prior art, the present invention has the following advantages: The α-glucosidase inhibitory peptide RGF and FLP provided by this invention have the effect of inhibiting α-glucosidase. Glucosidase has a significant inhibitory effect (IC50). 50 The concentrations were 0.599 mg / mL and 1.344 mg / mL, respectively, which are superior to the peptides (GEY(IC)) reported in existing literature. 50 (2.70 mg / mL), GYG (IC) 50 (1.50 mg / mL) and PFP (IC) 50 (3.10 mg / mL) indicates that it has good potential for adjuvant hypoglycemic applications.
[0021] This invention uses earthworms as raw material and employs an integrated autolysis-enzymatic hydrolysis-ultrafiltration process to obtain small molecule peptides <3kDa. The conditions are mild and the operation is simple. The obtained small molecule peptides are beneficial for inhibiting the activity of α-glucosidase in vivo.
[0022] This invention combines LC / MSMS technology with bioinformatics screening, utilizing a three-tiered screening strategy of Peptide Ranker activity prediction, molecular docking, and in vitro activity verification to efficiently identify the highly inhibitory small peptides Arg-Gly-Phe (RGF) and Phe-Leu-Pro (FLP) from earthworm enzymatic hydrolysates. Compared with traditional peptide screening methods, this method shortens the time to obtain target peptide sequences, significantly improves screening efficiency, and reduces research and development costs.
[0023] This invention not only provides a new approach for the high-value development and utilization of earthworms, but also provides new raw materials and technical support for the development of naturally sourced hypoglycemic functional foods, special medical foods and related medicines.
[0024] This invention not only fills the research gap in blood glucose-lowering active peptides from earthworms and enriches the library of food-derived α-glucosidase inhibitory peptides, but also provides a scalable technical strategy for the targeted preparation and efficient screening of highly active blood glucose-lowering peptides from animal-derived matrices. Attached Figure Description
[0025] Figure 1 Earthworm source α in this embodiment of the invention Technical roadmap for screening glucosidase inhibitory peptides.
[0026] Figure 2 Different molecular weight components of earthworm protein hydrolysate affect α Effect of glucosidase inhibition activity.
[0027] Figure 3 α-synthetic polypeptide Graph showing the glucosidase inhibition rate.
[0028] Figure 4 For peptide RGF IC 50 Value results graph.
[0029] Figure 5 For peptide FLP IC 50 Value results graph.
[0030] Figure 6 For polypeptide RGF and α Three-dimensional structure diagram of glucosidase molecular docking.
[0031] Figure 7 For peptide FLP and α Three-dimensional structure diagram of glucosidase molecular docking. Detailed Implementation
[0032] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. For process parameters not specifically specified, conventional techniques can be referred to. In the following examples, the in vitro α-glucosidase assay method is as follows: Prepare a sample solution of a specific mass concentration. Accurately pipette 25 μL of α-glucosidase solution and mix it with 50 μL of sample solution, then incubate at 37.5 ℃ for 10 min. Next, add 50 μL of p-nitrophenyl glucoside (p-NPG, 5 mM) to the mixture and incubate at 37 ℃ for 30 min. The inhibitory activity of α-glucosidase is calculated using the following formula: =[1 (OD 样品 -OD 样品空白 ) / (OD 对照 -OD 对照空白 )]×100 The degree of hydrolysis was determined using the TNBS method, as follows: Take 1 mL of enzyme digest diluted to an appropriate concentration, add 1 mL of 0.1 mol / L phosphate buffer (pH=8.2) and 1 mL of 0.05% (w / v) TNBS solution, and react in a light-protected water bath at 50℃ for 1 h. Then add 2 mL of 0.1 M HCl and shake to terminate the reaction. Add 5 mL of deionized water, continue shaking to mix, and after the reaction liquid cools, determine A. 340 nm. Using L-leucine as a standard, a standard solution was prepared, and the free amino group concentration was determined according to the method described above. A standard curve was plotted with L-leucine concentration on the x-axis and A340 nm on the y-axis. The free amino group concentration in the sample was calculated based on the standard curve. The formula for calculating the degree of hydrolysis is as follows: DH = h / h tot ×100% The method for determining protein recovery is as follows: The protein content in the enzyme hydrolysate was determined according to the Kjeldahl method in the national standard GB5009.5-2016, and the protein content of the enzyme hydrolysate was calculated using the following formula. Recovery rate: Protein recovery rate of enzymatic hydrolysate (%) = Protein content in enzymatic hydrolysate / Protein content in earthworm slurry × 100% Figure 1 The earthworm α is shown in an embodiment of the present invention. Technical roadmap for screening glucosidase inhibitory peptides.
[0033] The preparation conditions of earthworm protein peptides were optimized using a single-factor method. Example 1 (1) Raw material processing: Soak fresh earthworms (Eisenia fetida) in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, and then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 3000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, hydrolyze for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 2 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 4000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, hydrolyze for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 3 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 5000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, hydrolyze for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 4 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, enzymatic hydrolysis for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 5 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: 7000 u / g flavor protease was added to the cooling liquid obtained in step (2), the pH value was controlled at 8.5 and the temperature at 50℃, and the enzymatic hydrolysis was carried out for 4 hours to inactivate the enzyme. The supernatant was collected after centrifugation at 8000 rpm for 20 min. This supernatant is the earthworm protease hydrolysate (EPH). Table 1 shows the effects of different enzyme dosages on α-glucosidase inhibition rate, protein recovery rate, and degree of hydrolysis.
[0034] As shown in Table 1, the EPH prepared under the conditions of Example 4 has a high α-glucosidase inhibition rate, protein recovery rate and degree of hydrolysis.
[0035] Example 6 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, hydrolyze for 2 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 7 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, hydrolyze for 3 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 8 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, enzymatic hydrolysis for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 9 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, hydrolyze for 5 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Table 2 shows the effects of different enzymatic hydrolysis times on α-glucosidase inhibition rate, protein recovery rate, and degree of hydrolysis.
[0036] As shown in Table 2, the EPH prepared under the conditions of Example 8 has the highest α-glucosidase inhibition rate. Although its protein recovery rate and degree of hydrolysis are slightly lower than those of Example 9, there is no significant difference (P<0.05). Considering the time cost, the preferred enzymatic hydrolysis time is 4 h.
[0037] Example 10 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 45℃, hydrolyze for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 11 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, enzymatic hydrolysis for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 12 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 55℃, enzymatic hydrolysis for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 13 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 60℃, hydrolyze for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Table 3 shows the effects of different enzymatic hydrolysis temperatures on α-glucosidase inhibition rate, protein recovery rate, and degree of hydrolysis during secondary enzymatic hydrolysis.
[0038] As shown in Table 3, EPH prepared at an enzymatic hydrolysis temperature of 50-55 ℃ has a high α-glucosidase inhibition rate. Considering that the protein recovery rate is higher at an enzymatic hydrolysis temperature of 50 ℃, the preparation conditions of Example 11 are the optimal enzymatic hydrolysis temperature.
[0039] Example 14 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 7.5 and the temperature at 50℃, enzymatic hydrolysis for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 15 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 8.5 and the temperature at 50℃, enzymatic hydrolysis for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 16 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 9.5 and the temperature at 50℃, hydrolyze for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Example 17 (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain them. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at -40℃; (2) Preparation of autolysed hydrolysate: Earthworm sample and ultrapure water were mixed at a mass ratio of 1:2 to prepare earthworm slurry (33.3%, w / w). The autolysed hydrolysate was carried out under magnetic stirring at 40 °C for 2 h, followed by enzyme inactivation in a boiling water bath for 10 min. After the reaction solution cooled to room temperature, exogenous enzyme hydrolysis was continued. (3) Secondary enzymatic hydrolysis: Add 6000 u / g flavor protease to the cooling liquid obtained in step (2), control the pH value at 10.5 and the temperature at 50℃, hydrolyze for 4 hours, inactivate the enzyme, centrifuge at 8000 rpm for 20 min, collect the supernatant, which is the earthworm protease hydrolysate (EPH). Table 4 shows the effects of different enzymatic hydrolysis pH on α-glucosidase inhibition rate, protein recovery rate, and degree of hydrolysis.
[0040] As shown in Table 4, EPH prepared under enzymatic hydrolysis pH of 9.5-10.5 has a high α-glucosidase inhibition rate. Considering that the protein recovery rate is higher when the enzymatic hydrolysis pH is 9.5, the preparation conditions of Example 16 are the optimal enzymatic hydrolysis pH.
[0041] Example 18: EPH fractionation and screening of α-glucosidase inhibitory peptides (1) Ultrafiltration fractionation: The earthworm protein hydrolysate (EPH) obtained in Example 16 was centrifuged, and the α-glucosidase activity was verified by using an ultrafiltration membrane to retain three different molecular weight fractions with molecular weight cutoffs of <3 kDa, 3-10 kDa, and >10 kDa. Figure 2 The α-glucosidase half-maximal inhibitory concentration (IC50) of the ultrafiltration fraction with a molecular weight <3 kDa was 0.84 mg / mL, which was significantly lower than that of the 3-10 kDa (1.988 mg / mL) fraction and the >10 kDa (2.344 mg / mL) fraction. The <3 kDa fraction was freeze-dried. (2) LC MS / MS identification of peptide sequences: The <3kDa earthworm active peptide crude product obtained in step (1) after freeze-drying was reconstituted in ultrapure water at a ratio of 1:20 (g / mL), filtered through a 0.22μm microporous membrane, and identified by nano-LC-MS / MS; Chromatographic conditions: the pre-column was a PEPMAP NEO C18 (300 μm × 5 mm), the analytical column was a Reprosil-Pur 120 C18-AQ reversed-phase column (150 μm.d. × 170 mm, packing pore size 1.9 μm), the mobile phase A was ultrapure water containing 0.1% (v / v) formic acid (FA), the mobile phase B was an aqueous solution of 80% (v / v) acetonitrile (ACN) containing 0.1% (v / v) formic acid (FA), the flow rate was 600 nL / min, the analysis time was 66 min, and the elution method was gradient elution: 0 The amino acid sequences of the potential bioactive peptides were obtained by analyzing the data at the following concentrations: 4% (v / v) B at 2 min, 8% (v / v) B at 2 min, 28% (v / v) B at 35 min, 40% (v / v) B at 55 min, 95% (v / v) B at 56 min, and 95% (v / v) B at 66 min.
[0042] (3) Bioinformatics tools to assist in screening earthworm α Glucosidase inhibitory peptide: A total of 10,353 sequences were obtained through peptide profiling in step (2), of which 143 had a 10lgP>20 and a PeptideRanker score exceeding 0.9. Subsequently, based on the compositional characteristics of α-glucosidase inhibitory peptides: ① C-terminus Arg(R) / Lys(K) / Pro(P); ② N-terminus containing Leu(L) or at least one hydroxyl group or basic side chain (Ser(S) / Thr(T) / Tyr(Y) / Lys(K) / Arg(R) / His(H)); ③ Pro located at the penultimate position from the N-terminus to the C-terminus, potential active peptides were screened. Based on these three characteristics, 54 peptide sequences were selected for molecular docking.
[0043] (5) Molecular docking virtual screening of earthworm α Glucosidase inhibitory peptide: The candidate active peptides were docked with the α-glucosidase protein with PDB ID 3A4J using AutodockVina molecular docking software. The docking score (Vina score) was calculated, and peptide sequences with a Vina score < -8.9 were screened for synthesis and verification.
[0044] Six peptide sequences with binding energies below -8.9 kcal / mol were obtained through molecular docking. The six peptide sequences screened were: FEWP, WFFGHP, PGEFPW, RGF, FLP, and SPGEFPW. The potential earthworm α-glucosidase inhibitory peptide sequences and related properties are shown in Table 5.
[0045] Table 5. Sequences and related properties of potential earthworm α-glucosidase inhibitory peptides screened.
[0046] Example 19: Chemical Synthesis and Activity Verification of the Active Sequence The peptide sequences FEWP, WFFGHP, PGEFPW, RGF, FLP, and SPGEFPW obtained through virtual screening from the above enzymatic hydrolysates were synthesized in solid phase using the Fmoc amino acid solid-phase synthesis method by Nanjing Jietai Biotechnology Co., Ltd. In vitro α-glucosidase inhibitory activity was then verified, and their half-maximal inhibitory concentration (IC50) was determined. 50 ), The α-glucosidase inhibitory activities of synthetic peptides FEWP, WFFGHP, PGEFPW, RGF, FLP, and SPGEFPW, such as... Figure 3 As shown, among the above six synthetic peptides at 1 mg / mL, RGF and FLP exhibited good α-glucosidase inhibitory activity, such as... Figure 4-5 The IC is measured as shown. 50 The concentrations were 0.599 mg / ml and 1.344 mg / ml, respectively, to obtain α-glucosidase inhibitory peptides RGF and FLP.
[0047] Figure 6 The peptide RGF and α were shown. Three-dimensional structure diagram of glucosidase molecular docking.
[0048] Figure 7 The peptide FLP and α were shown. Three-dimensional structure diagram of glucosidase molecular docking.
[0049] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. An α-glucosidase inhibitory peptide derived from earthworms, characterized in that, The amino acid sequence of the α-glucosidase inhibitory peptide is Arg-Gly-Phe or Phe-Leu-Pro; Arg-Gly-Phe is abbreviated as RGF, and Phe-Leu-Pro is abbreviated as FLP.
2. The earthworm-derived α-glucosidase inhibitory peptide according to claim 1, characterized in that, The amino acid sequence was obtained from earthworm protein hydrolysate EPH using virtual screening technology.
3. The earthworm-derived α-glucosidase inhibitory peptide according to claim 2, characterized in that, The earthworm protein hydrolysate EPH was prepared by the following steps: (1) Raw material processing: Soak fresh earthworms in physiological saline to remove sand, then wash them with clean water and drain. Use a pulping machine to homogenize the fresh earthworms, then package them and store them at low temperature; (2) Autolytic enzymatic hydrolysis: Add water to the earthworm homogenate, stir to carry out pre-enzymatic hydrolysis, and terminate the reaction by boiling water bath after the pre-enzymatic hydrolysis is completed and then cool. (3) Secondary enzymatic hydrolysis: After the autolytic enzymatic hydrolysis is completed, flavor protease is added to the system, the pH and temperature of the enzymatic hydrolysis system are adjusted, and secondary enzymatic hydrolysis is carried out under stirring conditions; after the enzymatic hydrolysis reaction is completed, the reaction is terminated by boiling water bath and cooled. Centrifugation is used to collect the supernatant, which is the earthworm protein hydrolysate EPH.
4. The earthworm-derived α-glucosidase inhibitory peptide according to claim 3, characterized in that, In step (2), the mass ratio of the earthworm homogenate to water is 1:1 to 1:
4.
5. The earthworm-derived α-glucosidase inhibitory peptide according to claim 3, characterized in that, In step (3), the pH and temperature of the enzymatic hydrolysis system are adjusted, with the pH value being 7.5-10.5 and the temperature being 45-60℃.
6. The earthworm-derived α-glucosidase inhibitory peptide according to claim 3, characterized in that, In step (3), the amount of flavor protease added is 3000 u / g-7000 u / g based on protein content, and the enzymatic hydrolysis time is 2-5 hours.
7. The earthworm-derived α-glucosidase inhibitory peptide according to claim 2, characterized in that, The virtual screening technology includes the following steps: (1) Centrifuge the earthworm protein hydrolysate with EPH, use an ultrafiltration membrane to retain different molecular weight fractions to verify α-glucosidase activity, and freeze-dry the fraction with the best α-glucosidase activity inhibition effect. (2) The peptide sequence of the component with the best inhibitory effect on α-glucosidase activity was identified, and the amino acid sequence of the potential active peptide was obtained by database retrieval and analysis; (3) Screen peptides with 10lgP>20, score the potential active peptide sequences obtained, screen peptide sequences with scores>0.9, and then screen potential active peptides based on the amino acid composition characteristics of α-glucosidase inhibitory peptides. (4) The candidate active peptides were docked with the α-glucosidase protein with PDB ID 3A4J. The docking score Vina score was calculated and peptide sequences with Vina score < -8.9 were screened. (5) The peptide sequences obtained by screening were subjected to in vitro α-glucosidase inhibitory activity verification and their half-maximal inhibitory concentration was determined.
8. The earthworm-derived α-glucosidase inhibitory peptide according to claim 1, characterized in that, The α-glucosidase inhibitory peptide derived from earthworms can be artificially synthesized according to the amino acid sequence. The artificial synthesis includes synthesis using solid-phase synthesis methods, including Fmoc-SPPS, BOC-SPPS, and fragment condensation linking methods.
9. The use of the earthworm-derived α-glucosidase inhibitory peptide of claim 1 in the preparation of adjunctive hypoglycemic products.
10. The application according to claim 9, characterized in that, The preparation of the blood sugar-lowering product includes using α-glucosidase inhibitory peptide as an active ingredient in the preparation of foods, special medical purpose formula foods, or pharmaceuticals that help lower blood sugar.