Pearl mussel hypoglycemic polypeptide and application thereof

Abstract

The invention discloses a hyriopsis cumingii hypoglycemic polypeptide and application thereof. The amino acid sequence of the hyriopsis cumingii hypoglycemic polypeptide is as shown in SEQ ID NO.01. The IC50 value of the hyriopsis cumingii hypoglycemic polypeptide to alpha-glucosidase is 0.293 mmol / L, the IC50 value of the hyriopsis cumingii hypoglycemic polypeptide to dipeptidyl peptidase-IV is 34.78 mu mol / L, postprandial blood sugar rise can be effectively inhibited, insulin secretion is promoted, and the hyriopsis cumingii hypoglycemic polypeptide is suitable for adjuvant therapy and blood sugar management of type II diabetes patients.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a blood sugar-lowering polypeptide from pearl mussels and its applications. Background Technology

[0002] Diabetes mellitus is a common chronic metabolic disorder characterized by persistently elevated blood glucose levels. It is mainly classified into type I and type II, with type II diabetes accounting for the largest proportion. The disease is often caused by insufficient insulin secretion or insulin resistance, leading to an imbalance in the body's blood glucose regulation. Studies show that postprandial hyperglycemia is a typical early symptom of diabetes. Alpha-glucosidase, a key enzyme in the small intestine, catalyzes the breakdown of carbohydrates into glucose, promoting its absorption. Inhibiting alpha-glucosidase activity can slow down carbohydrate digestion, reduce postprandial blood glucose peaks, and thus effectively control the progression of diabetes. Furthermore, dipeptidyl peptidase-IV (DPP-IV) is a serine protease that rapidly degrades incretins such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). These hormones are crucial in stimulating insulin secretion and inhibiting glucagon release; once degraded by DPP-IV, they lower insulin levels and exacerbate blood glucose elevation. Therefore, DPP-IV inhibitors, by protecting the integrity of GLP-1 and GIP, can enhance insulin secretion and maintain blood glucose homeostasis.

[0003] Commonly used hypoglycemic drugs in clinical practice include alpha-glucosidase inhibitors such as acarbose and DPP-IV inhibitors such as sitagliptin. These drugs are mostly chemically synthesized and can effectively regulate blood sugar in the short term, but long-term use is often accompanied by significant side effects. For example, acarbose may cause gastrointestinal reactions such as bloating, diarrhea, and increased intestinal gas; sitagliptin and others may increase the risk of hypoglycemia and potentially damage liver and kidney function. These limitations have prompted research to shift towards safer natural alternatives. Bioactive peptides from food sources, as an emerging option, show significant advantages: low toxicity, few side effects, high bioavailability, and easy absorption. These peptides can exert hypoglycemic effects by competitively inhibiting enzyme activity or regulating related metabolic pathways, making them ideal components for adjunctive treatment of diabetes. In recent years, bioactive peptides obtained through enzymatic hydrolysis from various animal and plant proteins have been shown to have the potential to inhibit alpha-glucosidase and DPP-IV. These peptides often have simple structures and stable activity, making them easy to develop into functional foods or dietary supplements.

[0004] Pearl mussels are freshwater bivalve mollusks widely distributed in rivers and lakes of my country. They grow rapidly, reproduce rapidly, and are highly adaptable, making them primarily used for freshwater pearl farming. However, in the pearl production process, companies typically only extract the pearls, discarding or using byproducts such as mussel meat at low value, such as as animal feed, leading to significant resource waste. Pearl mussel meat is highly nutritious, especially rich in protein, accounting for over 50% of its dry weight, and is rich in essential amino acids and potentially bioactive components. If these proteins can be processed using appropriate technologies to transform them into high-value-added products, it can not only improve economic efficiency but also achieve sustainable resource utilization. Enzymatic hydrolysis is an effective method for extracting bioactive peptides from proteins. By selecting suitable proteases, large protein molecules can be degraded into smaller peptide fragments, which may retain or enhance specific biological activities, such as hypoglycemic effects.

[0005] In current technologies, the utilization of pearl mussel meat is mainly limited to simple extraction or feed processing, lacking optimized enzymatic hydrolysis processes targeting hypoglycemic activity. Enzymatic hydrolysis parameters such as enzyme type, pH, temperature, enzyme dosage, and time directly affect peptide yield and activity. Single-step enzymatic hydrolysis often fails to fully release active peptides, while multi-step hydrolysis can increase the degree of hydrolysis and generate more low-molecular-weight components. These low-molecular-weight peptides can be further enriched using separation and purification techniques such as ultrafiltration, gel chromatography, and high-performance liquid chromatography, and their sequences can be identified using De novo sequencing and bioinformatics tools. However, existing methods still have shortcomings in optimizing the specific enzymatic hydrolysis of pearl mussel meat, especially in the activity screening and purification pathway targeting dual-target inhibition of α-glucosidase and DPP-IV. This results in the purity and yield of active peptides failing to meet industrial requirements, impacting the value-added potential of pearl mussel processing byproducts. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects of the prior art and provide a pearl mussel hypoglycemic polypeptide.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned pearl mussel hypoglycemic polypeptide.

[0008] Another object of the present invention is to provide the application of the above-mentioned pearl mussel hypoglycemic polypeptide.

[0009] The technical solution of the present invention is as follows:

[0010] A blood sugar-lowering polypeptide from pearl mussels, the amino acid sequence of which is shown in SEQ ID NO.01.

[0011] The preparation method of the above-mentioned pearl mussel hypoglycemic polypeptide includes the following steps:

[0012] (1) After cleaning and chopping the pearl clam meat, mix it with a buffer solution of pH=8-9 and homogenize it. Then add alkaline protease and carry out the first enzymatic hydrolysis at 45-55 ℃ for 2-3 h. Then stop the first enzymatic hydrolysis and cool to room temperature. Adjust the pH to neutral, add neutral protease, and carry out the second enzymatic hydrolysis at 45-55 ℃ for 2-3 h. Finally stop the first enzymatic hydrolysis and cool to room temperature to obtain crude enzymatic hydrolysate.

[0013] (2) The crude enzymatic hydrolysate obtained in step (1) is centrifuged and the supernatant is separated by ultrafiltration to obtain a component with < 3 kDa;

[0014] (3) The < 3 kDa component obtained in step (2) is separated by gel chromatography and reversed-phase high-performance liquid chromatography in sequence to obtain the final product.

[0015] In a preferred embodiment of the present invention, in step (1), the ratio of the pearl clam meat to the buffer solution is 1 g: 6 mL.

[0016] More preferably, in step (1), the amount of alkaline protease added is 0.7-1.0%.

[0017] More preferably, in step (1), the amount of neutral protease added is 0.5-0.8%.

[0018] In a preferred embodiment of the present invention, in step (3), the gel chromatography separation is performed using a Superdex™ Peptide 10 / 300 GL gel filter column.

[0019] More preferably, the eluent for the gel chromatography separation is ultrapure water, and the eluent flow rate is 0.3-0.5 mL / min.

[0020] In a preferred embodiment of the present invention, in step (3), the reversed-phase high-performance liquid chromatography separation is performed using ZORBAX SB-C. 18 The column proceeds.

[0021] More preferably, in the eluent of the high-performance liquid chromatography separation, mobile phase A is ultrapure water containing 0.1% trifluoroacetic acid, mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid, the flow rate is 0.5 mL / min, and the column temperature is 25℃.

[0022] The application of the above-mentioned pearl mussel hypoglycemic peptides in the preparation of hypoglycemic compositions.

[0023] The beneficial effects of this invention are:

[0024] 1. The IC50 of the pearl mussel hypoglycemic polypeptide of the present invention against α-glucosidase 50The value was 0.293 mmol / L, and the IC50 for dipeptidyl peptidase-IV was... 50 The value was 34.78 μmol / L, which can effectively inhibit postprandial blood glucose elevation and promote insulin secretion, and is suitable for adjunctive treatment and blood glucose management in patients with type II diabetes.

[0025] 2. Compared with chemically synthesized drugs, the pearl mussel hypoglycemic polypeptide of the present invention is derived from the enzymatic hydrolysis product of pearl mussel meat protein. It has low toxicity, few side effects, high biocompatibility, and is easily absorbed by the human body. It can be used as a dietary supplement or functional food ingredient, avoiding gastrointestinal disorders, hypoglycemia, or liver and kidney damage caused by long-term drug use.

[0026] 3. This invention transforms the pearl production byproduct of pearl oysters, namely, the meat of the oyster, into high-value-added active peptide products, thereby reducing the waste of processing byproduct resources, improving economic efficiency, and promoting the sustainable development of the freshwater shellfish industry, thus achieving a green and circular economy.

[0027] 4. This invention employs a two-step enzymatic hydrolysis (combination of alkaline protease and neutral protease), combined with an activity-guided purification pathway using ultrafiltration, gel separation chromatography, and reversed-phase high-performance liquid chromatography, to ensure high hydrolysis degree and product purity. The <3kDa component in the enzymatic hydrolysis product exhibits the highest activity.

[0028] 5. The pearl mussel hypoglycemic peptide of the present invention acts on both α-glucosidase and DPP-IV, synergistically regulating carbohydrate digestion and incretin degradation, enhancing the overall hypoglycemic effect, and providing more comprehensive biological activity than single-target inhibition.

[0029] 6. The preparation method of this invention has clearly defined parameters (such as pH, temperature, and enzyme dosage). Combined with De novo sequencing and BIOPEP screening, it ensures accurate sequence identification and provides technical support for the standardized production of bioactive peptides from aquatic animals.

[0030] 7. The pearl mussel hypoglycemic polypeptide of the present invention can be used to develop new functional foods, promote the innovation of biotechnology in the field of diabetes prevention, and lay the foundation for the active exploration of similar shellfish proteins, thereby enhancing the industry's influence. Attached Figure Description

[0031] Figure 1 To demonstrate the inhibitory activity of the ultrafiltration fraction of the pearl mussel meat enzymatic hydrolysate in Example 1 of the present invention on α-glucosidase (A) and DPP-IV (B).

[0032] Figure 2 To illustrate the gel chromatography elution profile and inhibitory activity against α-glucosidase and DPP-IV in Example 1 of this invention. Wherein: (A) Gel chromatogram and IC50 of α-glucosidase. 50 (B) IC50 value determination of DPP-IV;50 value.

[0033] Figure 3 To illustrate the RP-HPLC elution chromatogram and the inhibitory activity against α-glucosidase and DPP-IV in Example 1 of this invention. Wherein: (A) RP-HPLC chromatogram; (B) Detection of α-glucosidase and DPP-IV inhibitory activities of each component.

[0034] Figure 4 This is the mass spectrum of the blood sugar-lowering polypeptide from pearl mussels in Example 1 of the present invention.

[0035] Figure 5 The IC50 of the pearl mussel hypoglycemic polypeptide in Example 1 of this invention against α-glucosidase and DPP-IV is shown. 50 Among them, the IC50 of (A) α-glucosidase is... 50 Value; (B) IC of DPP-IV 50 value. Detailed Implementation

[0036] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0037] The specific methods for testing the hypoglycemic activity in the following embodiments are as follows:

[0038] (1) Determination of α-glucosidase inhibitory activity

[0039] Mix 50 μL of the test sample with 50 μL of α-glucosidase (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) solution (0.5 U / mL) and incubate at 37℃ for 15 min. Then add 50 μL of 2.5 mmol / L p-nitrophenyl-α-D-glucopyranoside (PNPG) as the substrate to the mixture, incubate at 37℃ for 15 min, and then stop the reaction by adding 0.1 mol / L Na2CO3. Measure the absorbance at 405 nm. Simultaneously, a sample blank group, a control group, and a control blank group were set up, and the inhibition rate was calculated according to the formula:

[0040]

[0041] In the formula, A1, A2, A3, and A4 represent the absorbance values ​​at 405 nm of the blank tube, blank control tube, inhibition tube, and inhibition control tube, respectively.

[0042] (2) Determination of DPP-Ⅳ inhibitory activity

[0043] The activity of DPP-Ⅳ was measured using Gly-Pro-MCA as the substrate, with a sample without the added DPP-Ⅳ inhibitory peptide serving as the blank group. 50 μL of 20 mmol / L Tris-HCl buffer (pH 8.0) (70 μL for the blank group and 65 μL for the control group), 5 μL of DPP-Ⅳ enzyme (diluted 500 times) (purchased from Sigma-Aldrich, USA), and 15 μL of the sample to be tested were added sequentially to a black ELISA plate and thoroughly mixed. The plate was incubated at 37 °C for 5 min, then 30 μL of 10 mmol / L Gly-Pro-MCA substrate solution was added, and incubation continued for another 15 min. The fluorescence intensity of the reaction product was immediately measured using an ELISA reader at an excitation wavelength of 380 nm and an emission wavelength of 450 nm. The inhibition rate was calculated using the formula:

[0044]

[0045] In the formula, F 实验 F 对照 F 空白 The fluorescence intensity represents the values ​​for the experimental group, control group, and blank group.

[0046] Example 1

[0047] (1) Enzymatic hydrolysis of pearl clam meat:

[0048] Weigh 500 g of pearl mussel meat, wash and crush the tissue, add 3 L of 20 mmol / L phosphate buffer (pH 8.0), add 0.8% alkaline protease, and hydrolyze in a water bath at 50℃ for 2 h. After hydrolysis, inactivate the enzyme in a boiling water bath for 15 min, and then rapidly cool to room temperature. Perform a second hydrolysis on the hydrolysate, adjusting the pH to 7.0 with 1 mol / L hydrochloric acid, adding 0.6% neutral protease, and hydrolyze in a water bath at 45℃ for 1.5 h. After hydrolysis, react in a boiling water bath for 15 min to inactivate the enzyme, obtaining the crude hydrolysate.

[0049] (2) Separation and purification of enzymatic hydrolysis products

[0050] After centrifuging the crude enzymatic hydrolysate obtained in step (1), the supernatant was collected and concentrated using a 3 kDa ultrafiltration membrane. The filtrate was collected, and the inner and outer solutions were freeze-dried separately. After reconstitution, the inhibitory activities against α-glucosidase and DPP-IV were measured. The results are as follows: Figure 1 As shown;

[0051] Fractions <3 kDa were filtered through a 0.22 μm filter membrane, and 1 mL (10 mg / mL) was loaded onto a Superdex™ Peptide 10 / 300 GL gel filter column. Ultrapure water was used as the eluent (flow rate 0.3 mL / min), and the absorbance at 220 nm was measured. Fractions F1 and F2 were collected, freeze-dried, reconstituted, and their activity was measured again. The results are as follows: Figure 2 As shown;

[0052] The F1 component was further separated by RP-HPLC using a ZORBAX SB-C column. 18 (4.6 × 250 mm). In the eluent, mobile phase A was ultrapure water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid. The flow rate was 0.5 mL / min, the column temperature was 25℃, and 20 μL of sample was loaded. After collecting each fraction, the activities of α-glucosidase and DPP-IV were measured. The fraction F1-c with higher hypoglycemic activity was obtained, and the results are as follows. Figure 3 As shown.

[0053] (3) Determination of the amino acid sequence of hypoglycemic peptides

[0054] Using De novo sequencing and BIOPEP screening, a pearl mussel hypoglycemic polypeptide with the amino acid sequence Val-Pro-Ile-Tyr (SEQ ID NO. 01: VPIY) and a molecular weight of 490.59 Da was obtained. Figure 4 As shown.

[0055] (4) Test of hypoglycemic activity

[0056] The hypoglycemic activity of the above pearl mussel hypoglycemic peptides was determined, and the results are as follows: Figure 5 As shown, the IC50 of α-glucosidase 50 The IC50 of DPP-IV was 0.293 mmol / L. 50 It is 34.78 μmol / L.

[0057] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A blood sugar-lowering polypeptide from pearl mussels, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

01.

2. The method for preparing the blood sugar-lowering polypeptide from pearl mussels according to claim 1, characterized in that: Includes the following steps: (1) After cleaning and chopping the pearl clam meat, mix it with a buffer solution of pH=8-9 and homogenize it. Then add alkaline protease and carry out the first enzymatic hydrolysis at 45-55 ℃ for 2-3 h. Then stop the first enzymatic hydrolysis and cool to room temperature. Adjust the pH to neutral, add neutral protease, and carry out the second enzymatic hydrolysis at 45-55 ℃ for 2-3 h. Finally stop the first enzymatic hydrolysis and cool to room temperature to obtain crude enzymatic hydrolysate. (2) The crude enzymatic hydrolysate obtained in step (1) is centrifuged and the supernatant is separated by ultrafiltration to obtain a component with < 3 kDa; (3) The < 3 kDa component obtained in step (2) is separated by gel chromatography and reversed-phase high-performance liquid chromatography in sequence to obtain the final product.

3. The preparation method according to claim 2, characterized in that: In step (1), the ratio of pearl clam meat to the buffer solution is 1 g: 6 mL.

4. The preparation method according to claim 3, characterized in that: In step (1), the amount of alkaline protease added is 0.7-1.0%.

5. The preparation method according to claim 4, characterized in that: In step (1), the amount of neutral protease added is 0.5-0.8%.

6. The preparation method according to any one of claims 1 to 5, characterized in that: In step (3), the gel chromatography separation is performed using a Superdex™ Peptide 10 / 300 GL gel filter column.

7. The preparation method according to claim 6, characterized in that: The eluent for the gel chromatography separation is ultrapure water, and the eluent flow rate is 0.3-0.5 mL / min.

8. The preparation method according to any one of claims 1 to 5, characterized in that: In step (3), the reversed-phase high-performance liquid chromatography separation is performed using ZORBAX SB-C. 18 The column proceeds.

9. The preparation method according to claim 8, characterized in that: In the eluent of the high-performance liquid chromatography separation, mobile phase A is ultrapure water containing 0.1% trifluoroacetic acid, mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid, the flow rate is 0.5 mL / min, and the column temperature is 25℃.

10. The use of the pearl mussel hypoglycemic polypeptide of claim 1 in the preparation of hypoglycemic compositions.

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