Egg yolk-derived hypoglycemic peptide, and preparation method and application thereof

Abstract

The application discloses an egg yolk-derived hypoglycemic peptide as well as a preparation method and application thereof, and belongs to the technical field of polypeptides. The amino acid sequence of the egg yolk-derived hypoglycemic peptide is YVIQEDR. The application identifies a novel hypoglycemic peptide YVIQEDR from defatted egg yolk powder hydrolysate by using a molecular docking technology. It is verified that the hypoglycemic activity of the peptide YVIQEDR is stronger than that of the defatted egg yolk powder hydrolysate. In addition, the YVIQEDR has the advantages of non-toxicity, high safety, strong activity and the like, can be used as an ideal hypoglycemic effective factor, can be widely used in the fields of food, health products, medicine and the like, and has great application value.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to an egg yolk-derived hypoglycemic peptide, its preparation method, and its application. Background Technology

[0002] Diabetes is a prevalent chronic metabolic disease, and prolonged hyperglycemia can lead to various complications, seriously endangering human health. Currently, diabetes treatment mainly relies on medication and insulin injections, but long-term use of these methods may cause a series of side effects. Therefore, developing safe and effective alternative therapies for lowering blood sugar has become a current research hotspot.

[0003] Carbohydrates are the primary energy source in the human diet, and their digestion process significantly impacts blood glucose regulation. The digestion of carbohydrates into free glucose primarily relies on the combined action of α-amylase and α-glucosidase. Carbohydrates are initially hydrolyzed into disaccharides and oligosaccharides by α-amylase. Upon reaching the brush border of the small intestine, α-glucosidase efficiently catalyzes the breakdown of α-1,4 and α-1,6 glycosidic bonds into absorbable glucose. Due to the directional action of the intestine, α-glucosidase is considered an ideal target for developing safe and effective hypoglycemic drugs. Inhibiting α-glucosidase activity can delay the conversion of polysaccharides and disaccharides into absorbable glucose, thereby reducing postprandial blood glucose spikes and helping to control blood glucose fluctuations. This has significant therapeutic effects on early-stage diabetes and patients with impaired glucose tolerance, and is also suitable for some patients with insulin resistance. Furthermore, an advantage of α-glucosidase inhibitors is that they do not stimulate insulin secretion, thus avoiding hypoglycemia, which is crucial for preventing blood glucose fluctuations and maintaining stable blood glucose throughout the day. Acarbose, one of the first α-glucosidase inhibitors introduced to the market in 1990, remains the most prescribed drug to date. However, common adverse reactions such as bloating, abdominal pain, and diarrhea limit the tolerability of long-term use. Therefore, the discovery of safe and effective α-glucosidase inhibitory peptides from natural food sources has attracted widespread attention. These bioactive peptides are derived from foods that have been safely consumed long-term, generally exhibit good biocompatibility, and are less likely to produce serious side effects.

[0004] In recent years, research on functional foods and natural bioactive peptides has gradually become a hot topic in the field of diabetes prevention and treatment. Food-derived functional peptides, released through enzymatic hydrolysis from natural food sources such as dairy products, soybeans, fish, and eggs, possess a wide range of biological activities. Egg yolks, as a nutrient-dense natural raw material, are rich in high-quality protein, and functional peptides can be released from them through appropriate enzymatic hydrolysis. However, current research on specific peptides with hypoglycemic activity in egg yolk protein hydrolysates is relatively limited, and a clear structure-function relationship and target mechanism study are lacking. Therefore, developing a functional peptide derived from egg yolks with significant hypoglycemic activity has become an urgent need for diabetes nutritional intervention and functional food development. Summary of the Invention

[0005] The purpose of this invention is to provide an egg yolk-derived hypoglycemic peptide, its preparation method, and its application, in order to solve the problems existing in the prior art. This invention utilizes molecular docking-based directional screening technology to obtain the highly efficient hypoglycemic peptide YVIQEDR from defatted egg yolk powder hydrolysate. It can be used in the food, health product, pharmaceutical, and animal feed industries, and has broad application prospects.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an egg yolk-derived hypoglycemic peptide, wherein the amino acid sequence of the egg yolk-derived hypoglycemic peptide is YVIQEDR.

[0008] The present invention also provides a method for preparing the egg yolk-derived hypoglycemic peptide, comprising the following steps:

[0009] The defatted egg yolk powder was added to water and hydrolyzed using a single protease. After hydrolysis, the enzyme was inactivated, cooled, and the supernatant was collected by centrifugation to obtain the defatted egg yolk powder hydrolysate.

[0010] The defatted egg yolk powder hydrolysate was separated into peptides by HPLC.

[0011] The peptide was molecularly simulated and docked with α-glucosidase to screen and identify the egg yolk-derived hypoglycemic peptide YVIQEDR.

[0012] Optionally, the single protease includes papain, alkaline protease, trypsin, or neutral protease.

[0013] Preferably, the single protease is trypsin.

[0014] Optionally, the amount of enzyme added for enzymatic hydrolysis is 2000 U / g, and the hydrolysis time is 2 hours.

[0015] Optionally, the centrifugation is performed at 4°C and 8000 rpm for 15 minutes.

[0016] Optionally, the mobile phase A of the HPLC liquid chromatography is 0.1% formic acid, and the mobile phase B is a mixture of 0.1% formic acid and 99.9% acetonitrile.

[0017] The present invention also provides the application of the egg yolk-derived hypoglycemic peptide in the preparation of hypoglycemic drugs.

[0018] The present invention also provides the application of the egg yolk-derived hypoglycemic peptide in the preparation of health products that help maintain healthy blood sugar levels.

[0019] The present invention also provides a hypoglycemic drug comprising the aforementioned egg yolk-derived hypoglycemic peptide.

[0020] The present invention also provides a health product that helps maintain healthy blood sugar levels, comprising the aforementioned egg yolk-derived hypoglycemic peptide.

[0021] The present invention discloses the following technical effects:

[0022] This invention utilizes molecular docking technology to identify a novel hypoglycemic peptide, YVIQEDR, from defatted egg yolk powder hydrolysate. Verification showed that YVIQEDR exhibits stronger hypoglycemic activity compared to defatted egg yolk powder hydrolysate. Furthermore, YVIQEDR possesses advantages such as being non-toxic, highly safe, and highly active, making it an ideal and effective hypoglycemic agent with significant application value in the food, health product, and pharmaceutical industries. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is the secondary mass spectrum of the peptide YVIQEDR;

[0025] Figure 2 This is a schematic diagram of the docking of the polypeptide YVIQEDR with the α-glucosidase molecule;

[0026] Figure 3 for Figure 2 The interaction between the black-framed polypeptide YVIQEDR and α-glucosidase; the green dashed lines represent hydrogen bonds, and the arcs represent residues involved in hydrophobic interactions.

[0027] Figure 4 To evaluate the in vitro hypoglycemic activity of the polypeptide YVIQEDR. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example 1: Preparation and Screening of Polypeptides

[0034] 1. Enzymatic hydrolysis

[0035] Using defatted egg yolk powder (purchased from Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd., Baiyunshan Pharmaceutical Factory) as raw material, double-distilled water was added at a volume ratio of powder to water of 1:10. Enzymatic hydrolysis was performed using a single protease (including papain, alkaline protease, trypsin, and neutral protease; trypsin was used in this example). The enzyme dosage was 2000 U / g, and the pH of the solution was adjusted to 8 (the suitable pH for the enzyme). After mixing, the solution was placed in a constant temperature shaking water bath at 37°C for 2 hours. During the hydrolysis, the pH was adjusted to 8 every hour. After hydrolysis, the hydrolysate was boiled in a water bath for 10 minutes to inactivate the enzyme, then cooled to room temperature and centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant (i.e., the defatted egg yolk powder hydrolysate) was collected and stored for later use.

[0036] 2. Peptide identification

[0037] The amino acid sequences of peptides in defatted egg yolk powder hydrolysate were identified using LC-MS / MS.

[0038] The hydrolysate was separated using a nanoElute HPLC system with a flow rate of nanoliters. Column information: Thermo Scientific EASY column, 25 cm, ID 75 μm, 1.9 μm, C18; Mobile phase A: 0.1% formic acid; Mobile phase B: a mixture of 0.1% formic acid and 99.9% acetonitrile (ACN); Flow rate: 300 nL / min; Liquid phase gradient over 30 min: 0 min–18 min, linear gradient of phase B from 5% to 35%; 18 min–20 min, linear gradient of phase B from 35% to 80%; 20 min–30 min, phase B maintained at 80%.

[0039] Samples were separated by chromatography and then analyzed by mass spectrometry using a timsTOFPro mass spectrometer. The ion source voltage was set to 1.5 kV, and both MS and MSMS were detected and analyzed using TOF. The mass spectrometry scan range was set to 100 m / z–1700 m / z. The data acquisition mode was Parallel Accumulation Serial Fragmentation (PASEF). After primary mass spectrometry acquisition, precursor ions were acquired 8 times in PASEF mode with a cycle window of 0.95 s, and secondary spectra were obtained for charge numbers in the range of 0–5. The dynamic exclusion time for tandem mass spectrometry was set to 24 s to avoid duplicate precursor ions.

[0040] 3. Peptide screening: Select peptides with a molecular weight of less than 1 kDa, a length of 2-10 amino acid residues, non-toxic and with good water solubility from the numerous matched peptide sequences.

[0041] 4. Molecular docking

[0042] Molecular docking was performed using AutoDock software, and the specific steps are as follows:

[0043] First, the receptor α-glucosidase was prepared. The α-glucosidase molecule was obtained from the RCSB database (3WY1). Water molecules and small ligand molecules were removed, and hydrogen and charge were added. Then, the ligands were prepared. The 3D structure of the screened peptides was drawn using an online peptide structure generator (https: / / cloud.yinfotek.com / console / ). Molecular docking was performed using Auto Dock Vina. The center coordinates of the active pocket were set as x = -6.665, y = -12.514, z = 21.514, and the docking box size was set to 80×80×80. The binding energy between the peptide and α-glucosidase was obtained. The peptide YVIQEDR (Tyr-Val-Ile-Gln-Glu-Asp-Arg) showed the best docking performance, with a binding energy of -8.4 kcal / mol. The secondary mass spectrum of YVIQEDR is shown below. Figure 1 A schematic diagram of its docking with α-glucosidase molecules is shown below. Figure 2 Interactions Figure 3 ,Depend on Figure 2-3 It is known that the polypeptide YVIQEDR interacts with α-glucosidase through hydrogen bonds, hydrophobic interactions, and salt bridges.

[0044] Example 2: Determination of the hypoglycemic activity of active peptides

[0045] The selected peptide YVIQEDR was synthesized in vitro at a concentration of 2 mg / mL. The hypoglycemic activity of the peptide was evaluated using the defatted egg yolk powder hydrolysate prepared in Example 1 as a control.

[0046] Method for determining α-glucosidase inhibition rate: Both the substrate p-nitrophenylglucopyranoside and α-glucosidase were dissolved in phosphate buffer (0.1 M, pH 6.8). The sample was mixed thoroughly with 0.5 U / mL α-glucosidase and incubated at 37°C for 15 min. 2.5 mM substrate solution was added to the mixture, and incubation continued at 37°C for 20 min. Finally, 1 M Na₂CO₃ was added to terminate the reaction. The control group used phosphate buffer instead of the sample solution, and the blank group used phosphate buffer instead of the enzyme solution. The absorbance was measured at 405 nm and calculated using the following formula.

[0047]

[0048] Where: C is the absorbance value without sample, A is the absorbance value with sample, and B is the blank control of A.

[0049] Results of hypoglycemic activity are shown in Figure 4The results showed that 2 mg / mL YVIQEDR inhibited α-glucosidase by 64.36%, which was significantly higher than that of defatted egg yolk powder hydrolysate (37.56%, P<0.05), thus verifying the hypoglycemic effect of YVIQEDR.

[0050] Based on the above results, YVIQEDR can interact with α-glucosidase through hydrogen bonds, hydrophobic interactions, and salt bridges, thereby inhibiting α-glucosidase activity and exerting a hypoglycemic effect.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of egg yolk-derived hypoglycemic peptides in the preparation of hypoglycemic drugs, characterized in that, The amino acid sequence of the egg yolk-derived hypoglycemic peptide is YVIQEDR.

2. The application of egg yolk-derived hypoglycemic peptides in the preparation of health products that help maintain healthy blood sugar levels, characterized in that... The amino acid sequence of the egg yolk-derived hypoglycemic peptide is YVIQEDR.

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