Eggshell membrane hydrolyzate and peptide for lowering blood sugar and preparation method and application thereof

Abstract

The present invention discloses an eggshell membrane hydrolyzate, a peptide and a preparation method and application thereof for lowering blood sugar, and relates to the technical field of functional protein hydrolyzates. The eggshell membrane hydrolyzate provided by the present invention is prepared by enzymolyzing eggshell membranes with alkaline protease and papain, and then inactivating the enzymes to obtain a primary enzymolyzate. The present invention also provides a digestion product that also has a blood sugar lowering effect by simulating gastrointestinal digestion. The present invention also obtains a peptide segment by ultrafiltration screening of the primary enzymolyzate. These eggshell membrane hydrolyzates, digestion products and peptide segments can be used to prepare blood sugar lowering drugs or foods with auxiliary blood sugar lowering function, or to measure blood sugar lowering effects for purposes other than disease diagnosis and treatment.

Description

Technical Field

[0001] The invention relates to the technical field of functional protein hydrolysates, and in particular to eggshell membrane hydrolysates and peptides for lowering blood sugar, and preparation methods and applications thereof. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar levels, which is mainly divided into type I, type II and gestational type. Type II diabetes is a non-communicable endogenous disease with impaired or absent insulin signaling. Patients with type II diabetes account for more than 90% of the total number of diabetes patients. Since 2010, the number of patients with type II diabetes has increased year by year worldwide and is expected to reach 1.31 billion by 2050. The number of people with diabetes in China accounts for a quarter of the total population of China, so the treatment of diabetes is urgent.

[0003] At present, the main drugs for the treatment of type II diabetes are the following categories: biguanides, thioureas, glinides, thiazolidinediones, α-glucosidase inhibitors, sodium-glucose co-transporter 2 inhibitors, and DPP-IV inhibitors. However, drug treatment is often accompanied by certain side effects. Compared with other types of drugs, DPP-IV inhibitors have fewer side effects, except that they cannot be used by some patients with acute nephritis and acute pancreatitis. They will not cause digestive system problems such as gastrointestinal bloating like α-glucosidase inhibitors, nor will they cause certain cardiovascular diseases due to water and sodium retention like insulin, rosiglitazone and pioglitazone. The hypoglycemic mechanism of DPP-IV inhibitory peptides is to inhibit the degradation of glucagon-like peptide-1 by DPP-IV enzymes in the human body, and then enable glucagon-like peptide-1 to secrete insulin normally, thereby maintaining the balance of blood sugar in the body.

[0004] Compared with the above drugs, protein peptides have fewer side effects; the metabolic end product is amino acids, which are relatively safe and avoid the disadvantage of immunogenicity compared to protein macromolecules. Eggshell membrane, as a by-product of egg processing, contains a variety of proteins such as collagen and keratin, as well as bioactive substances such as hyaluronic acid and chondroitin sulfate, and is a potential source of active peptides. Therefore, if a hypoglycemic peptide can be developed from eggshell membrane as a DPP-IV inhibitor, it will not only expand the processing chain of animal-derived foods vertically, but also greatly enhance the high-value utilization of livestock and poultry by-products, which is of great significance. Summary of the invention

[0005] The present invention provides eggshell membrane hydrolyzate and peptide for reducing blood sugar, and a preparation method and application thereof, which are specifically achieved through the following technologies.

[0006] A method for preparing an eggshell membrane hydrolysate comprises enzymolyzing the eggshell membrane using alkaline protease and papain, and obtaining a primary enzymolyzate after inactivating the enzymes to prepare the eggshell membrane hydrolysate; the ratio of the enzyme specific activities of the alkaline protease and the papain is (1-3):1, and the total enzyme specific activity of the alkaline protease and the papain is 5-10 U / mg based on the mass of the eggshell membrane.

[0007] The eggshell membrane hydrolyzate is used for preparing a blood sugar lowering drug or a food with an auxiliary blood sugar lowering function, or for measuring the blood sugar lowering effect for purposes other than disease diagnosis and treatment.

[0008] It should be noted that the eggshell membrane hydrolysate provided by the present invention, in addition to being able to be used to prepare hypoglycemic drugs and functional foods, the above-mentioned "determination of hypoglycemic effects for purposes other than disease diagnosis and treatment" generally refers to use in laboratories and testing institutions as a reagent for constructing corresponding animal models, conducting scientific research, or as a comparative evaluation of hypoglycemic effects, as well as other unlisted application methods that are not used for the diagnosis or treatment of diabetes or hyperglycemia.

[0009] Furthermore, the preparation method also includes a pretreatment process of the eggshell membrane, specifically, steam explosion of the eggshell membrane or grinding of the eggshell membrane into powder, and then mixing with water to prepare an eggshell membrane suspension.

[0010] Generally, for water-insoluble solid film-shaped substances composed of proteins in plants and animals, in order to facilitate subsequent processing, in some implementation cases, the film-shaped substances can be processed by steam explosion. In other implementation cases, in order to achieve the same purpose, the film-shaped substances can also be directly ground into fine powder and then mixed with water.

[0011] The eggshell membrane used in the present invention can be directly purchased from the market as a finished eggshell membrane, or can be directly taken from fresh eggshells and obtained after the egg white and mucus are washed off.

[0012] Optionally, in some implementation cases, the steam explosion treatment method is to maintain the pressure at 1.0-2.0 MPa for 1-5 min.

[0013] Optionally, in other implementation cases, the eggshell membrane is dried, crushed, and sieved to obtain the product. For example, the eggshell membrane is dried in an oven at 50-70° C., crushed with a crusher, and sieved through a 100-200 mesh sieve to obtain the product; it can generally be stored in a dark and ventilated place.

[0014] By comparison, the method of treating eggshell membranes with steam explosion can promote the mixing effect of eggshell membranes and water, improve the enzymatic hydrolysis efficiency and the yield of eggshell membrane hydrolysate. The method of crushing eggshell membranes into fine powder can also obtain the eggshell membrane hydrolysate of the present invention, but the enzymatic hydrolysis efficiency and the yield of eggshell membrane hydrolysate will be slightly affected.

[0015] In the preparation method provided by the present invention, when alkaline protease and papain are used for enzymolysis, it is necessary to select the enzymolysis conditions (pH value, temperature, etc.) of the two proteases and co-enzymolyze the eggshell membrane under the condition that both have good enzymolysis activity.

[0016] Furthermore, the enzymatic hydrolysis method is: pH 5.5-8.5, 37-60°C for 2-6h.

[0017] Through in vitro activity verification of the liquid eggshell membrane hydrolysate (primary enzymatic hydrolysate) prepared by the above method, it was found that the eggshell membrane hydrolysate has a good DPP-IV inhibition rate and can be directly made into a blood sugar-lowering drug, or made into a food (health food) with auxiliary blood sugar-lowering function. The prepared dosage forms include but are not limited to capsules, tablets, oral preparations and the like.

[0018] Furthermore, the primary enzymatic hydrolysate is subjected to ultrafiltration treatment in the following three ways respectively, and at least one of the three ultrafiltration products is used as the eggshell membrane hydrolysate:

[0019] (1) passing the primary enzymatic hydrolysate through an ultrafiltration membrane with a pore size of 3 kDa, and taking the first filtrate;

[0020] (2) passing the primary enzymatic hydrolysate through an ultrafiltration membrane with a pore size of 3 kDa, passing the first retentate through an ultrafiltration membrane with a pore size of 10 kDa, and taking the second filtrate;

[0021] (3) The primary enzymatic hydrolysate is passed through an ultrafiltration membrane with a pore size of 10 kDa to obtain a second retentate.

[0022] After studying the DPP-IV inhibition rate of the primary enzymatic hydrolysate, the primary enzymatic hydrolysate was further subjected to ultrafiltration treatment, specifically using ultrafiltration membranes with pore sizes of 3kDa and 10kDa. Specifically using the above method, three different ultrafiltration products were obtained, namely, three ultrafiltration products with molecular weight <3kDa, 3kDa≤molecular weight≤10kDa and molecular weight>10kDa. The study found that the DPP-IV inhibition rate of these three ultrafiltration products was also very high, and the DPP-IV inhibition rate of the ultrafiltration products with molecular weight <3kDa and 3kDa≤molecular weight≤10kDa even exceeded that of the primary enzymatic hydrolysate. Similarly, mixing the above three ultrafiltration products in pairs can also be used as eggshell membrane hydrolysates for corresponding applications.

[0023] Furthermore, the eggshell membranes are further hydrolyzed with pepsin and trypsin in sequence to obtain a final hydrolyzate as the eggshell membrane hydrolyzate.

[0024] In order to verify the DPP-IV inhibition rate of the primary hydrolysate and the ultrafiltration products after the above ultrafiltration treatment after being taken by patients and digested by the gastrointestinal tract, the gastrointestinal digestion process of the primary hydrolysate and the three ultrafiltration products was simulated. The test results showed that the digestion products of the primary hydrolysate and the digestion products after the three ultrafiltrations also had a high DPP-IV inhibition rate after simulated digestion.

[0025] Optionally, in some embodiments, when further enzymatic hydrolysis is performed using pepsin, the amount of pepsin used is 2000-5000 U / mg based on the eggshell membrane raw material.

[0026] Optionally, in other embodiments, when trypsin is used for further enzymatic hydrolysis, the amount of trypsin used is 100-500 U / mg based on the eggshell membrane raw material.

[0027] The eggshell membrane hydrolyzate prepared by the preparation method of the present invention can be directly made into a liquid preparation product, or can be concentrated for easy storage and application. Furthermore, it can be freeze-dried to finally prepare eggshell membrane hydrolyzate powder.

[0028] The present invention also provides an eggshell membrane hydrolyzate, which is prepared by any one of the above preparation methods.

[0029] The present invention also provides a peptide segment, which is used to prepare a blood sugar-lowering drug or a food with auxiliary blood sugar-lowering function, or to measure the blood sugar-lowering effect for the purpose of non-disease diagnosis and treatment. The amino acid sequence of the peptide segment is shown in SEQ ID NO.1.

[0030] The ultrafiltration product with the best DPP-IV inhibition rate and a molecular weight of <3kDa was selected for HPLC-MS / MS identification and bioinformatics analysis, that is, the peptide property calculator online tool was used to predict the function of the peptide; then AutoDock Vina was used for molecular simulation docking identification and final sequencing, and a single peptide with high activity was obtained. The amino acid sequence of the single peptide is shown in SEQ ID NO.1, specifically Gly-Pro-Pro-His-Phe-Leu-Pro-Phe.

[0031] This single peptide was synthesized by solid phase synthesis and its DPP-IV inhibitory activity was measured in vitro, which confirmed that this peptide fragment has a certain DPP-IV inhibitory ability and can provide a new treatment option for patients with type 2 diabetes.

[0032] The present invention also claims to protect the above-mentioned eggshell membrane hydrolyzate or peptide fragment, and its use in preparing blood sugar lowering drugs or foods with auxiliary blood sugar lowering function, or in measuring blood sugar lowering effects for purposes other than disease diagnosis and treatment.

[0033] The present invention also provides a substance, characterized in that the substance is any one of the following:

[0034] (1) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.1;

[0035] (2) a plasmid vector containing the nucleic acid molecule;

[0036] (3) A recombinant cell comprising the nucleic acid molecule or plasmid vector.

[0037] Those skilled in the art will appreciate that the term "nucleic acid molecule" in the present application actually includes any one or both of the complementary double strands. The nucleotide sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0038] Those skilled in the art will appreciate that the term "plasmid vector" in this application refers to a sequence that can effectively express the peptide segment shown in SEQ ID NO.1. The plasmid vector may include an optional control sequence. The control sequence is operably linked to the nucleic acid molecule. Among them, the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in the host. The vector plasmid (such as a vector or a transformant) thus constructed can effectively express the above-mentioned peptide segment.

[0039] When the nucleic acid molecule is connected to the vector plasmid, the nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. The nucleic acid molecule is operably connected to the control element.

[0040] According to an embodiment of the present invention, a vector plasmid may refer to a cloning vector or an expression vector, which may be obtained by operably connecting a nucleic acid molecule to a commercially available vector (such as a plasmid or a viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids may be used, such as pSeTag2, PEE14, pMH3, etc.

[0041] Those skilled in the art will understand that the term "recombinant cell" in this application refers to a cell containing the above nucleic acid molecule or the above vector plasmid. The cell may be a prokaryotic cell, a eukaryotic cell or a bacteriophage. Further, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc.; the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian bacterium, etc.

[0042] Compared with the prior art, the present invention is beneficial in that: the present invention provides an eggshell membrane hydrolyzate prepared by enzymolysis of eggshell membrane with alkaline protease and papain, and further provides a peptide segment. The eggshell membrane hydrolyzate and peptide segment have good DPP-IV inhibitory activity and can provide a new treatment option for patients with type II diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the mass spectrometry identification diagram of the peptide segment in Example 4;

[0044] Figure 2 is the DPP-IV activity inhibition rate of the primary enzymatic hydrolysate and three ultrafiltration products;

[0045] Figure 3 is the DPP-IV activity inhibition rate of the primary enzymatic hydrolysate and three ultrafiltration products after the digestion treatment of Example 3;

[0046] Figure 4 It is the DPP-IV activity inhibition rate of the peptide synthesized in Example 4. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] In the following implementation cases, eggshell membranes are enzymatically hydrolyzed using alkaline protease and papain, and the enzymes are inactivated to obtain a primary enzymatic hydrolyzate, that is, an eggshell membrane hydrolyzate.

[0049] The ratio of the enzyme specific activities of the alkaline protease and papain is (1-3):1, and the total enzyme specific activity of the alkaline protease and papain is 5-10 U / mg based on the mass of the eggshell membrane.

[0050] The alkaline protease used in the following implementation cases was purchased from Wuhan Feiyang Biological Co., Ltd., model: 200 U / mg, and the papain was purchased from Shanghai Yuanye Biotechnology Co., Ltd., model: 800 U / mg.

[0051] Accordingly, in the following examples, the specific activity of alkaline protease is 6 U / mg based on the mass of the eggshell membrane, and the specific activity of papain is 3 U / mg based on the mass of the eggshell membrane.

[0052] In some implementation cases, the eggshell membrane raw material used is prepared by taking the eggshell membrane of fresh eggs, washing off the mucus and egg white, and drying it.

[0053] Furthermore, in order to improve the subsequent enzymatic hydrolysis efficiency, the eggshell membranes are subjected to steam explosion treatment or the eggshell membranes are ground into powder and then water is added to prepare an eggshell membrane suspension.

[0054] In some implementation cases, the eggshell membrane suspension is prepared by: (1) steam-exploding the eggshell membrane at a pressure of 1.0-2.0 MPa for 1-5 min, and drying at low temperature after the treatment; or, grinding the eggshell membrane into powder using a grinder and passing it through a 120-mesh sieve to obtain eggshell membrane powder; (2) stirring and mixing the steam-exploded eggshell membrane or eggshell membrane powder with pure water in a mass volume ratio (w / v) of 1:(18-20), and leaving it overnight to prepare an eggshell membrane suspension.

[0055] In some implementation cases, in order to increase the degree of swelling and solubility of eggshell membranes in water, promote the rapid enzymatic hydrolysis of alkaline protease and papain, and increase the yield of eggshell membrane hydrolysates, steam explosion treatment is generally used to pretreat the eggshell membranes.

[0056] When using alkaline protease and papain for enzymolysis, it is necessary to select the two proteases under conditions where both have good enzymolysis activity to enzymolyze the eggshell membranes together according to the enzymolysis conditions (pH value, temperature, etc.). In some implementation cases, the enzymolysis conditions are generally pH = 5.5-8.5, and enzymolysis at 37-60°C for 2-6 hours.

[0057] In some implementation cases, the primary enzymatic hydrolysate is further subjected to any of the following ultrafiltration treatments:

[0058] (1) passing the primary enzymatic hydrolysate through an ultrafiltration membrane with a pore size of 3 kDa, and taking a first filtrate; the molecular weight of the first filtrate is less than 3 kDa;

[0059] (2) passing the primary enzymatic hydrolysate through an ultrafiltration membrane with a pore size of 3 kDa, passing the first retentate through an ultrafiltration membrane with a pore size of 10 kDa, and taking a second filtrate; the molecular weight of the first retentate is between 3 kDa and 10 kDa;

[0060] (3) The primary enzymatic hydrolysate is passed through an ultrafiltration membrane with a pore size of 10 kDa to obtain a second retentate having a molecular weight of >10 kDa.

[0061] Example 1: Preparation of primary enzymatic hydrolysate

[0062] The preparation method of the eggshell membrane hydrolyzate of this embodiment is as follows:

[0063] (1) steam explosion the cleaned eggshell membranes and then dry them at 60° C.

[0064] (2) placing the dried eggshell membranes in a container, stirring and hydrating them with purified water at a weight-to-volume ratio (w / v) of 1:20 overnight to obtain an eggshell membrane suspension;

[0065] (3) transferring the eggshell membrane suspension to an enzymatic hydrolysis tank, adjusting the enzymatic hydrolysis conditions to pH = 8.0, 55° C., adding alkaline protease (4 mg / g) and papain (6 mg / g) for enzymatic hydrolysis for 6 h, and obtaining a primary enzymatic hydrolyzate;

[0066] (4) Freeze-drying to prepare primary enzymatic hydrolysate powder.

[0067] Example 2: Preparation of ultrafiltration product

[0068] The three ultrafiltration products were prepared by the following methods:

[0069] (1) The primary enzymatic hydrolysate prepared in Example 1 was passed through an ultrafiltration membrane with a pore size of 3 kDa to obtain a first filtrate with a molecular weight of less than 3 kDa, which was prepared into a freeze-dried powder for later use.

[0070] (2) The primary enzymatic hydrolysate is first passed through an ultrafiltration membrane with a pore size of 3 kDa, and the first retentate is passed through an ultrafiltration membrane with a pore size of 10 kDa. The second filtrate is taken, and the molecular weight is 3 kDa ≤ ≤ 10 kDa, and is prepared into a freeze-dried powder for later use.

[0071] (3) The primary enzymatic hydrolysate is passed through an ultrafiltration membrane with a pore size of 10 kDa, and the second retentate with a molecular weight of >10 kDa is obtained and prepared into a freeze-dried powder for later use.

[0072] Example 3: Preparation of the final enzymatic hydrolysate

[0073] 1. Preparation of final enzymolysis product using the primary enzymolysis product of Example 1

[0074] (1) Simulate gastric digestion process:

[0075] Take 10 ml of the 10 mg / mL primary enzymatic hydrolysate solution prepared in Example 1, adjust and maintain the pH value of the reaction system at 3.0 and the temperature at 37°C, add 1.6 ml of 25000 U / ml porcine pepsin (purchased from Shanghai Yuanye Biotechnology Co., Ltd., 30 U / mg) solution, and continue magnetic stirring for 120 min to obtain a primary digestion product.

[0076] Optionally, the dosage of porcine pepsin is: the enzyme specific activity is 2000-5000U / mg based on the mass of eggshell membrane.

[0077] In this embodiment, the specific dosage of porcine pepsin is as follows: the enzyme specific activity is 2000 U / mg based on the volume of the primary enzymatic hydrolysate solution.

[0078] (2) Simulating the intestinal digestion process

[0079] Take 10 ml of the primary digestion product, adjust and maintain the pH value of the reaction system at 7.0 and the temperature at 37°C, add 2.5 mL of 800 U / mL trypsin (purchased from Shanghai Yuanye Biotechnology Co., Ltd., 250 U / mg) solution, and continue magnetic stirring for 120 min to obtain the secondary digestion product, i.e., the final enzymatic hydrolysate, which is lyophilized into powder for standby use.

[0080] Optionally, the amount of trypsin used is: the enzyme specific activity is 100-500 U / mg based on the volume of the primary digestion product.

[0081] In this embodiment, the specific dosage of trypsin is: the enzyme specific activity is 100 U / mg based on the volume of the primary digestion product.

[0082] 2. Preparation of the final enzymatic hydrolysate using the three ultrafiltration products of Example 2

[0083] The final enzymatic hydrolysate was prepared using the same method as above with the three ultrafiltration products.

[0084] Example 4: Screening, identification and synthesis of target peptides

[0085] The ultrafiltration product with a molecular weight of less than 3 kDa in Example 2 was subjected to HPLC-MS / MS. The mobile phase of the liquid chromatography method includes mobile phase A and mobile phase B; the elution column is a domestic fused silica capillary column (ID 75 μm, 150 mm, Upchurch, Oak Harbor, WA) containing C-18 resin (300A, 5 μm, Varian, Lexington, MA). Mobile phase A is 0.1% formic acid. Mobile phase B is 0.1% trifluoroacetic acid. The elution program is: 0-8 min is 3% B, 8-40 min is 3-50% B, 40-55 min is 50% B, 55-75 min is 50-99% B, 75-85 min is 99-3% B, the flow rate is 0.3 μL / min, and the injection volume is 1 μL.

[0086] Mass spectrometry identification results Figure 1As shown; bioinformatics analysis was performed, that is, the peptide property calculator online tool was used to predict the function of the peptide, and then AutoDock Vina was used for molecular simulation docking, and the sequence of the single peptide with high DPPV-IV inhibitory activity was identified as shown in SEQ ID NO.1, specifically:

[0087] Gly-Pro-Pro-His-Phe-Leu-Pro-Phe.

[0088] The peptide Gly-Pro-Pro-His-Phe-Leu-Pro-Phe was synthesized by solid phase synthesis, and the purity was analyzed by HPLC, and then lyophilized powder was prepared for use.

[0089] Test Example 1: In vitro DPPV-IV activity verification

[0090] The DPP-IV inhibition rate of the samples was determined using a DPP-IV inhibition screening kit (fluorescence method, purchased from Cayman, No. 700210). The samples tested were the primary hydrolysate prepared in Example 1, the three ultrafiltration products prepared in Example 2, the four final hydrolysates prepared in Example 3, and the peptides in Example 4, and sitagliptin (1 mM, 10 μL) was selected as a positive control.

[0091] The determination method is:

[0092] (1) Before the assay, the sample lyophilized powder was dissolved in a buffer solution (20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl and 1 mM EDTA) and diluted to prepare a sample solution (10 mg / mL). 10 μL of the sample solution and 50 μL of the DPP-IV solution were added to a 96-well plate, mixed, and incubated at 37°C for 10 min.

[0093] (2) Add 25 μL of substrate solution to all wells and incubate at 37°C for 15 min;

[0094] (3) Use an enzyme marker to perform fluorescence detection under the conditions of excitation wavelength of 360 nm and emission wavelength of 460 nm and record the results, which are recorded as F 样品 and F 对照 .

[0095] All of the above steps (1) to (3) are carried out in a dark environment to ensure that the substrate is not decomposed.

[0096] (4) The DPP-IV activity inhibition rate was calculated according to the following formula: DPP-IV inhibition rate = [(F 样品 -F 对照 ) / F 对照]×100%.

[0097] The results of DPP-IV activity inhibition rate are as follows Figure 2-4 shown.

[0098] Figure 2 The DPP-IV activity inhibition rates of the primary hydrolysate and three ultrafiltration products. In the figure, the "Control" group is the positive control group (sitagliptin); the "hydrolysate" is the primary hydrolysate, and "<3kDa" is the ultrafiltration product with a molecular weight of <3kDa. Figure 2 It can be seen that under the premise that the DPP-IV inhibition rate of sitagliptin is 100%, the primary hydrolysate can exceed 60% of the relative activity of DPP-IV inhibition rate; among the three ultrafiltration products, the DPP-IV inhibition rates of the ultrafiltration products with molecular weight <3kDa, 3kDa≤molecular weight≤10kDa and molecular weight>10kDa decrease in turn. More importantly, the DPP-IV inhibition rate of the ultrafiltration product with molecular weight <3kDa is significantly higher than that of the primary hydrolysate, with a relative activity of 75%, and the ultrafiltration product with 3kDa≤molecular weight≤10kDa is also higher than the primary hydrolysate, and the ultrafiltration product with molecular weight>10kDa is only slightly lower than the ultrafiltration product.

[0099] Figure 3 is the DPP-IV activity inhibition rate of the primary enzymatic hydrolysate and three ultrafiltration products after the digestion treatment of Example 3. Figure 3 It can be seen that the DPP-IV activity inhibition rate of the four samples has decreased significantly; however, the primary enzymatic hydrolysate still reaches 45%; the molecular weight <3kDa still has nearly 60% activity, 3kDa≤molecular weight≤10kDa still has nearly 55% activity, and the molecular weight >10kDa still has 50% activity. This shows that although it has been digested, the high DPP-IV activity inhibition rate still makes these digestion products still have considerable application prospects.

[0100] Figure 4 The DPP-IV activity inhibition rates of the undigested primary enzymatic hydrolysate, the ultrafiltration product with a molecular weight of <3 kDa, and the peptide synthesized in Example 4. Figure 4 It can be seen that the DPP-IV activity inhibition rate of the synthetic peptide segment reached more than 80%, compared with more than 60% of the primary hydrolysate and 75% of the ultrafiltration product. This shows that the peptide segment screened in Example 4 has a very high DPP-IV inhibition ability and can be used in the treatment of type II diabetes.

[0101] It should be noted that in order to reduce the effect of gastrointestinal proteases on the activity of eggshell membrane hydrolysates or peptides after oral administration, the eggshell membrane hydrolysates or peptides can be loaded and embedded with commonly used drug delivery carriers on the market to accelerate their direct intestinal absorption. In view of the huge demand for oral administration in current clinical use, various new technologies have emerged that can overcome the gastrointestinal barrier of peptides, such as enteric coatings, enzyme inhibitors, permeation enhancers, nanoparticles, and intestinal microdevices. Some new technologies have been developed in clinical trials and are even on the market.

[0102] For example, in some embodiments, pepsin can easily cleave a variety of proteins or peptides in an acidic environment, but when the pH value is greater than 3, pepsin begins to lose its activity. Therefore, if we can adjust the pH value of the microenvironment to above 5, we can prevent the peptides from being degraded in the stomach.

[0103] In some embodiments, enteric coating is often used to overcome the degradation of PP in the stomach. Some organic acids, such as citric acid, are often used as pH lowering agents to inhibit the activity of intestinal enzymes.

[0104] Enzyme inhibitors inactivate target enzymes by reversibly or irreversibly binding to specific sites of the enzymes. Many chemical molecules can inhibit the activity of enzymes, such as bile acid and its derivative diisopropyl fluorophosphate. Peptide and modified peptide-derived enzyme inhibitors have been widely studied, such as aprotinin for inhibiting trypsin and chymotrypsin and soybean trypsin for inhibiting pancreatic endopeptidase. Chicken and duck egg mucins are recently developed and are considered safer. They can effectively inhibit the activity of α-chymotrypsin and trypsin and provide 100% protection for insulin. Therefore, in some embodiments, this type of enzyme inhibitor can also be used to inhibit the effects of gastrointestinal proteases on the peptides of the present invention.

[0105] Compared with the stomach and small intestine, the colon is a more suitable absorption site because of the reduced enzyme activity and neutral pH. In addition, the colon has a longer residence time and is more responsive to absorption enhancers. Therefore, the colon is an ideal administration site for orally active peptides.

[0106] In addition, by encapsulation, active peptides can be protected from hydrolysis and enzymatic degradation in the harsh gastric environment of the gastrointestinal tract to improve their intestinal absorption. In addition to protecting active peptides from degradation, particles can also enhance transepithelial transport. For example, polymer nanoparticles (such as polyethylene glycol), lipid nanoparticles (such as silica nanoparticles, gold nanoparticles), liposomes, nanoemulsions and inorganic nanoparticles can be used for encapsulation.

[0107] In summary, there are currently many technical methods that can prepare the eggshell membrane hydrolyzate or peptide provided by the present invention into drugs or foods that are easy to take, easy to absorb, and have unaffected DPP-IV inhibitory activity.

[0108] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A peptide segment, characterized in that: The peptide segment is used to prepare a blood sugar lowering drug or a food with auxiliary blood sugar lowering function, and the amino acid sequence of the peptide segment is shown in SEQ ID NO.

1.

2. A substance, characterized in that The substance is any of the following: (1) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.1; (2) a plasmid vector containing the nucleic acid molecule; (3) A recombinant cell comprising the nucleic acid molecule or plasmid vector.

3. Use of the peptide segment according to claim 1 in the preparation of a blood sugar lowering drug or a food having an auxiliary blood sugar lowering function.

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