CN118272477B

CN118272477B - Eggshell membrane hydrolyzate and peptide for lowering blood sugar and preparation method and application thereof

Info

Publication number: CN118272477B
Application number: CN202410504920.0A
Authority: CN
Inventors: 黄茜; 王莎莎; 宁彤; 刘美玉
Original assignee: Huazhong Agricultural University
Current assignee: Anhui Rongda Food Co ltd
Priority date: 2024-04-25
Filing date: 2024-04-25
Publication date: 2025-01-10
Anticipated expiration: 2044-04-25
Also published as: CN118272477A

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

Eggshell membrane hydrolysate and peptide for reducing blood sugar, and preparation method and application thereof

Technical Field

The invention relates to the technical field of functional protein hydrolysates, in particular to eggshell membrane hydrolysates for reducing blood sugar, peptides, and a preparation method and application thereof.

Background

Diabetes is a metabolic disease characterized by hyperglycemia, and is largely classified into type I, type II, and gestational. Type II diabetes is a non-infectious endogenous disease in which insulin signals are impaired or absent, and patients with type II diabetes account for more than 90% of the total number of patients with diabetes. The number of worldwide type II diabetics increases year by year since 2010, with the estimated 13.1 billion being reached in 2050. The number of people suffering from diabetes in China is one fourth of the total number of people in China, so that the treatment of diabetes is not slow.

The current drugs for treating type II diabetes mainly comprise biguanides, thioureas, glinide, thiazolidinediones, alpha-glucosidase inhibitors, sodium-glucose cotransporter 2 inhibitors and DPP-IV inhibitors. However, the drug treatment is often accompanied by certain side effects, and compared with other drugs, DPP-IV inhibitors have smaller side effects except that some patients with acute nephritis and acute pancreatitis can not use the DPP-IV inhibitors, and the DPP-IV inhibitors can not cause digestive system problems such as flatulence and the like the alpha-glucosidase inhibitors, and can not cause certain cardiovascular diseases such as insulin, rosiglitazone, pioglitazone and the like caused by sodium retention. The hypoglycemic mechanism of DPP-IV inhibiting peptide is to inhibit the degradation of glucagon-like peptide-1 by DPP-IV enzyme in human body and then make glucagon-like peptide-1 secrete insulin normally, so as to maintain the blood sugar balance in human body.

Compared with the medicines, the protein peptide has smaller side effect, the metabolic end product is amino acid, the safety is higher, and the disadvantage of immunogenicity is avoided compared with protein macromolecules. Eggshell membrane is a by-product of egg processing, contains various proteins such as collagen and keratin, and also contains bioactive substances such as hyaluronic acid and chondroitin sulfate, and is a potential active peptide source. Therefore, if the hypoglycemic peptide can be developed from eggshell membranes to serve as the DPP-IV inhibitor, the processing chain of animal-derived foods can be longitudinally expanded, and the high-value utilization of livestock and poultry byproducts can be greatly improved, so that the hypoglycemic peptide has very important significance.

Disclosure of Invention

The invention provides eggshell membrane hydrolysate and peptide for reducing blood sugar, and a preparation method and application thereof, which are realized by the following technology.

The eggshell membrane hydrolysate is prepared by carrying out enzymolysis on eggshell membrane by using alkaline protease and papain, and inactivating enzyme to obtain primary enzymolysis product, wherein the specific activity ratio of the alkaline protease to the papain is 1 to 1, and the total specific activity of the alkaline protease and the papain is 5-10U/mg based on the mass of the eggshell membrane.

The eggshell membrane hydrolysate is used for preparing a hypoglycemic drug or food with an auxiliary hypoglycemic function, or measuring the hypoglycemic effect for the purposes of non-disease diagnosis and treatment.

It should be noted that, in addition to being able to be used for preparing hypoglycemic drugs and functional foods, the above-mentioned "measuring hypoglycemic effect for non-disease diagnosis and treatment" generally refers to a method of performing scientific research in a laboratory or a detection mechanism as a reagent for constructing a corresponding animal model or for performing comparative evaluation of hypoglycemic effect, and other application methods not listed and not used for diagnosis or treatment of diabetes or hyperglycemia diseases.

Further, the preparation method also comprises a pretreatment process of the eggshell membrane, specifically steam blasting the eggshell membrane or grinding the eggshell membrane, and then mixing the eggshell membrane with water to prepare an eggshell membrane suspension.

In general, for film-shaped solid substances composed of proteins in water-insoluble animals and plants, the film substances may be treated by steam explosion in some embodiments in order to facilitate the subsequent treatment. In other embodiments, the film material may be ground directly to a fine powder and then mixed with water for the same purpose.

The eggshell membrane selected by the invention can be directly purchased into a finished product of the eggshell membrane on the market, and can be obtained by directly taking off fresh eggshells and cleaning egg white and mucus.

Alternatively, in some embodiments, the steam explosion treatment is performed at a pressure of 1.0-2.0MPa for 1-5min.

Optionally, in other embodiments, the eggshell membrane is dried, crushed, and sieved. For example, oven drying at 50-70deg.C, pulverizing with crusher, sieving with 100-200 mesh sieve, and storing in a dark place.

Through comparison, the mode of treating the eggshell membrane by steam explosion can promote the mixing effect of the eggshell membrane and water, and the enzymolysis efficiency and the yield of eggshell membrane hydrolysate are improved. The eggshell membrane hydrolysate of the invention can be obtained by crushing eggshell membrane into micro powder, but the enzymolysis efficiency and the yield of the eggshell membrane hydrolysate can be slightly influenced.

In the preparation method provided by the invention, when alkaline protease and papain are used for enzymolysis, the eggshell membrane is required to be subjected to enzymolysis under the conditions (pH value, temperature and the like) of the two proteases, and the two proteases are selected to be subjected to enzymolysis under the condition of good enzymolysis activity.

Further, the enzymolysis method is that the enzymolysis is carried out for 2 to 6 hours at the pH of 5.5 to 8.5,37 to 60 ℃.

After the in vitro activity verification of the liquid eggshell membrane hydrolysate (primary zymolyte) prepared by the method, the eggshell membrane hydrolysate has better DPP-IV inhibition rate, can be directly prepared into a medicament for reducing blood sugar or prepared into food (health food) with the auxiliary blood sugar reducing function, and the prepared dosage forms comprise but are not limited to capsules, tablets, oral administration agents and the like.

Further, the primary zymolyte is respectively subjected to ultrafiltration treatment in the following 3 ways, and at least one of the obtained 3 ultrafiltration products is used as the eggshell membrane hydrolysate:

(1) Passing the primary zymolyte through an ultrafiltration membrane with the pore diameter of 3kDa, and taking a first filtrate;

(2) Passing the primary zymolyte through an ultrafiltration membrane with the aperture of 3kDa, passing the first retentate through an ultrafiltration membrane with the aperture of 10kDa, and taking a second filtrate;

(3) The primary zymolyte was passed through an ultrafiltration membrane with a pore size of 10kDa and the second retentate was taken.

After the DPP-IV inhibition of the primary substrate was studied, the primary substrate was further subjected to ultrafiltration treatment using ultrafiltration membranes with pore sizes of 3kDa and 10 kDa. By adopting the method, 3 different ultrafiltration products are obtained, namely three ultrafiltration products with molecular weight less than 3kDa, molecular weight less than or equal to 3kDa and less than or equal to 10kDa and molecular weight more than 10kDa in sequence. The DPP-IV inhibition rate of the 3 ultrafiltration products is also very high, and the DPP-IV inhibition rate of the ultrafiltration products with the molecular weight of <3kDa and the molecular weight of less than or equal to 3kDa and less than or equal to 10kDa is even higher than that of the primary zymolyte. Similarly, the above three ultrafiltration products can be mixed two by two to be used as eggshell membrane hydrolysates correspondingly.

Further, pepsin and trypsin are further adopted for enzymolysis treatment in sequence, and the final enzymolysis product is obtained and is used as eggshell membrane hydrolysate.

In order to verify the DPP-IV inhibition rate of the primary zymolyte and the ultrafiltration product after ultrafiltration treatment after being taken by a patient, the primary zymolyte and 3 ultrafiltration products respectively simulate the gastrointestinal digestion process. The test results show that the digestion products of the primary zymolyte after simulated digestion and the digestion products after 3 kinds of ultrafiltration also have higher DPP-IV inhibition rate.

Optionally, in some embodiments, pepsin is used in an amount of 2000-5000U/mg based on eggshell membrane material when further enzymatically treated with pepsin.

Alternatively, in other embodiments, trypsin is used in an amount of 100-500U/mg based on eggshell membrane material when further enzymatic treatment is performed with trypsin.

The eggshell membrane hydrolysate prepared by the preparation method can be directly prepared into a liquid preparation product, and can be subjected to concentration treatment for convenient storage and application. Further, freeze-drying treatment can be carried out, and finally eggshell membrane hydrolysate powder is prepared.

The invention also provides an eggshell membrane hydrolysate which is prepared by adopting any one of the preparation methods.

The invention also provides a peptide segment, wherein the peptide segment is used for preparing a hypoglycemic drug or food with an auxiliary hypoglycemic function, or determining hypoglycemic effect for the purposes of non-disease diagnosis and treatment, and the amino acid sequence of the peptide segment is shown as SEQ ID NO. 1.

The ultra-filtration product with the best DPP-IV inhibition rate and the molecular weight of less than 3kDa is selected for HPLC-MS/MS identification and bioinformatics analysis, namely, the polypeptide is subjected to functional prediction by using peptide property calculator on-line tools, and then the polypeptide is subjected to molecular simulation docking identification and final sequencing by using AutoDock Vina, so that the single peptide with higher activity is obtained. The amino acid sequence of the single peptide is shown as SEQ ID NO.1, specifically Gly-Pro-Pro-His-Phe-Leu-Pro-Phe.

The single peptide is synthesized by using a solid phase synthesis mode, and DPP-IV inhibition activity of the single peptide is measured in vitro, so that the peptide has certain DPP-IV inhibition capability, and a new treatment option can be provided for type II diabetics.

The invention also claims application of the eggshell membrane hydrolysate or peptide fragment in preparing hypoglycemic drugs or foods with auxiliary hypoglycemic functions or in measuring hypoglycemic effects for non-disease diagnosis and treatment.

The invention also provides a substance, which is characterized by being any one of the following substances:

(1) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 1;

(2) A plasmid vector comprising the nucleic acid molecule;

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

It will be appreciated by those skilled in the art that the term "nucleic acid molecule" according to the present application actually includes either one or both of the complementary double strands. The nucleotide sequence in the present application includes a DNA form or an RNA form, and disclosure of one of them means that the other is also disclosed.

It will be appreciated by those skilled in the art that the term "plasmid vector" according to the present application refers to a sequence capable of efficiently expressing the peptide fragment shown in SEQ ID NO. 1. The plasmid vector may include optional control sequences. The control sequence is operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in the host. The vector plasmid (such as a vector or a transformant) thus constructed can efficiently express the above-mentioned peptide fragment.

In the case of ligating the above-mentioned nucleic acid molecule to a vector plasmid, the nucleic acid molecule may be directly or indirectly linked to control elements on an expression vector, as long as these control elements are capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. The nucleic acid molecule is operably linked to a control element.

According to embodiments of the invention, vector plasmids may be referred to as cloning vectors, as well as expression vectors, and may be obtained by operably linking a nucleic acid molecule to a commercially available vector (e.g., a plasmid or viral vector). The vector of the present invention is not particularly limited, and commonly used plasmids such as pSeTag, PEE14, pMH3, etc. can be used.

It will be understood by those skilled in the art that the term "recombinant cell" according to the present application refers to a cell comprising a nucleic acid molecule as described above or a vector plasmid as described above. The cell may be a prokaryotic cell, a eukaryotic cell or a phage. Further, the prokaryotic cells are colibacillus, bacillus subtilis, streptomycete or Proteus mirabilis, etc., the eukaryotic cells are fungi, insect cells, plant cells or mammalian bacteria, etc.,

Compared with the prior art, the invention has the beneficial effects that the eggshell membrane hydrolysate prepared by enzymolysis of eggshell membranes with alkaline protease and papain is provided, and a peptide fragment is further provided. The eggshell membrane hydrolysate and peptide fragment have good DPP-IV inhibitory activity, and can provide a new treatment choice for type II diabetics.

Drawings

FIG. 1 is a mass spectrum identification chart of the peptide fragment of example 4;

FIG. 2 shows the DPP-IV inhibition of primary enzyme and three ultrafiltration products;

FIG. 3 shows the DPP-IV inhibition of the primary enzyme and three ultrafiltration products from the digestion process of example 3;

FIG. 4 shows the DPP-IV activity inhibition ratio of the peptide synthesized in example 4.

Detailed Description

The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.

In the following embodiments, the eggshell membrane is subjected to enzymolysis by using alkaline protease and papain, and the primary zymolyte is obtained after enzyme deactivation, so that the eggshell membrane hydrolysate is obtained.

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

The alkaline protease used in the following examples was purchased from the company of Boehmeria of Wuhan, model number 200U/mg, and papain was purchased from the company of Shanghai Yes Biotechnology, model number 800U/mg.

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

In some embodiments, the eggshell membrane raw material is prepared by taking eggshell membrane of fresh egg, cleaning mucus and egg white, and drying.

Further, in order to improve the subsequent enzymolysis efficiency, the eggshell membrane is subjected to steam explosion treatment or ground into powder, and then water is added to prepare an eggshell membrane suspension.

In some embodiments, the eggshell membrane suspension is prepared by (1) performing steam explosion treatment on eggshell membrane under 1.0-2.0MPa for 1-5min, drying at low temperature, or grinding eggshell membrane into powder with a grinder and sieving with 120 mesh sieve to obtain eggshell membrane powder, (2) mixing the eggshell membrane or eggshell membrane powder subjected to steam explosion with purified water according to a mass-volume ratio (w/v) of 1 (18-20), and standing overnight to obtain eggshell membrane suspension.

In some embodiments, in order to promote the swelling and dissolution degree of eggshell membrane in water, promote the alkaline protease and papain to perform enzymolysis rapidly, promote the yield of eggshell membrane hydrolysate, and generally select a steam explosion treatment mode to pretreat eggshell membrane.

When alkaline protease and papain are used for enzymolysis, enzymolysis conditions (pH value, temperature and the like) of the two proteases are required to be selected for enzymolysis of eggshell membranes under the condition of good enzymolysis activity. In some embodiments, the enzymatic hydrolysis conditions are typically ph=5.5-8.5,37-60 ℃ for 2-6 hours.

In some embodiments, the primary substrate is further subjected to any one of the following ultrafiltration treatments:

(1) Passing the primary zymolyte through an ultrafiltration membrane with a pore size of 3kDa to obtain a first filtrate, wherein the molecular weight of the first filtrate is less than 3kDa;

(2) Passing the primary zymolyte through an ultrafiltration membrane with the aperture of 3kDa, passing the first retentate through an ultrafiltration membrane with the aperture of 10kDa, and taking a second filtrate, wherein the molecular weight of the first retentate is between 3kDa and 10 kDa;

(3) The primary zymolyte was passed through an ultrafiltration membrane with a pore size of 10kDa and the second retentate was taken. This second retentate has a molecular weight of >10kDa.

Example 1 preparation of Primary zymolyte

The preparation method of the eggshell membrane hydrolysate in this example is as follows:

(1) Performing steam explosion treatment on the cleaned eggshell membrane, and drying at a low temperature of 60 ℃;

(2) Placing the dried eggshell membrane into a container, and stirring and hydrating the eggshell membrane with purified water for overnight by using the mass-to-volume ratio (w/v) of 1:20 to obtain an eggshell membrane suspension;

(3) Transferring eggshell membrane suspension into an enzymolysis tank, adjusting enzymolysis conditions to be pH= 8.0,55 ℃, adding alkaline protease (4 mg/g) and papain (6 mg/g) for enzymolysis for 6 hours, and obtaining a primary enzymolysis product;

(4) And (5) performing low-temperature freeze drying to prepare primary zymolyte powder.

EXAMPLE 2 preparation of ultrafiltration product

The three ultrafiltration products are respectively prepared by the following methods:

(1) The primary zymolyte prepared in example 1 is passed through an ultrafiltration membrane with a pore size of 3kDa to obtain a first filtrate with a molecular weight of <3kDa, and is made into freeze-dried powder for later use.

(2) The primary zymolyte firstly passes through an ultrafiltration membrane with the aperture of 3kDa, the first trapped substance passes through an ultrafiltration membrane with the aperture of 10kDa, the second filtered substance is taken, the molecular weight of 3kDa is less than or equal to 10kDa, and freeze-dried powder is prepared for standby.

(3) The primary zymolyte passes through an ultrafiltration membrane with the aperture of 10kDa, the second trapped substance is taken, the molecular weight is more than 10kDa, and the freeze-dried powder is prepared for standby.

Example 3 preparation of final zymolyte

1. Preparation of final substrate Using the Primary substrate of example 1

(1) Simulating the gastric digestion process:

10mL of 10mg/mL of the primary hydrolysate solution prepared in example 1 was taken, the pH value of the reaction system was adjusted and maintained=3.0 and the temperature was 37 ℃, 1.6mL of 25000U/mL of porcine pepsin (purchased from Shanghai source leaf biotechnology Co., ltd., 30U/mg) solution was added, and magnetic stirring was continued for 120min, to obtain a primary digested product.

Optionally, the dosage of the pig pepsin is that the specific activity of the pig pepsin is 2000-5000U/mg based on the mass of eggshell membrane.

In the embodiment, the specific dosage of the pig pepsin is that the specific activity of the pig pepsin is 2000U/mg calculated by the volume of the primary zymolyte solution.

(2) Simulating intestinal digestion process

10ML of primary digestion product is taken, the pH value of the reaction system is regulated and kept to be 7.0 and the temperature is 37 ℃, 2.5mL of 800U/mL trypsin (purchased from Shanghai source leaf biotechnology Co., ltd., 250U/mg) solution is added, magnetic stirring is continued for 120min, and secondary digestion product, namely final zymolyte, is obtained, and the secondary digestion product is freeze-dried into powder for later use.

Alternatively, trypsin is used in an amount such that the specific activity of the enzyme is 100-500U/mg based on the volume of the primary digestion product.

In this example, trypsin was used in an amount such that the specific enzyme activity was 100U/mg based on the volume of the primary digestion product.

2. Preparation of final enzyme substrate Using the 3 ultrafiltration products of example 2

The final substrate was prepared from three ultrafiltration products using the same procedure as described above.

Example 4 screening, identification and Synthesis of target peptide fragments

The ultrafiltration product of example 2 with a molecular weight of <3kDa was subjected to HPLC-MS/MS. The mobile phase of the liquid chromatography comprises mobile phase A and mobile phase B, and the elution column is a domestic fused silica capillary column (ID 75 μm,150mm,Upchurch,Oak Harbor,WA) containing C-18 resin (300A, 5 μm, varian, lexington, mass.). Mobile phase a was 0.1% formic acid. The mobile phase B is 0.1% trifluoroacetic acid. The elution procedure is that the elution time is 0-8min of 3% B,8-40min of 3-50% B,40-55min of 50% B,55-75min of 50-99% B,75-85min of 99-3% B, the flow rate is 0.3 mu L/min, and the sample injection amount is 1 mu L.

The mass spectrum identification result is shown in figure 1, bioinformatics analysis is carried out, namely, peptide property calculator on-line tools are adopted to carry out functional prediction on the polypeptide, then AutoDock Vina is utilized to carry out molecular simulation docking, and the sequence of the high DPPV-IV inhibitory activity single peptide is identified as shown in SEQ ID NO.1, specifically:

Gly-Pro-Pro-His-Phe-Leu-Pro-Phe。

The synthetic peptide Gly-Pro-Pro-His-Phe-Leu-Pro-Phe is synthesized by solid phase synthesis, and HPLC purity analysis is performed to prepare freeze-dried powder for standby.

Test example 1 in vitro DPPV-IV Activity verification

The DPP-IV inhibition of the samples was determined using a DPP-IV inhibition screening kit (fluorescence, purchased from Cayman Co., no. 700210). The samples tested were the primary enzyme preparation of example 1, the three ultrafiltration products of example 2, the four final enzyme preparations of example 3, and the peptide fragment of example 4, respectively, and sitagliptin (1 mM, 10. Mu.L) was used as a positive control.

The measuring method comprises the following steps:

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

(2) To all wells 25 μl of substrate solution was added and incubated at 37 ℃ for 15min;

(3) Fluorescence detection was performed with an enzyme-labeled instrument at excitation wavelength of 360nm and emission wavelength of 460nm, and the results were recorded as F _{Sample of} and F _Control.

All of the above steps (1) - (3) are carried out in a dark environment to ensure that the substrate is not degraded.

(4) The DPP-IV activity inhibition rate is calculated according to the following formula, wherein the DPP-IV inhibition rate is= [ (F _{Sample of}-F_Control)/F_Control ]. Times.100%).

The DPP-IV activity inhibition results are shown in FIGS. 2-4.

FIG. 2 shows the DPP-IV inhibition of primary enzyme and three ultrafiltration products, wherein the "Control" group is the positive Control group (sitagliptin), the "enzyme" is the primary enzyme, and the "<3kDa" is the ultrafiltration product with a molecular weight of <3 kDa. As can be seen from FIG. 2, the primary substrate can have a DPP-IV inhibition ratio of more than 60% relative activity on the premise that the DPP-IV inhibition ratio of sitagliptin is 100%, and the DPP-IV inhibition ratio of the three ultrafiltration products, namely the ultrafiltration products with the molecular weight of <3kDa, the molecular weight of 3kDa less than or equal to 10kDa and the molecular weight of >10kDa, is sequentially reduced. More importantly, the DPP-IV inhibition rate of the ultrafiltration product with the molecular weight of <3kDa is obviously higher than that of the primary zymolyte, the relative activity reaches 75 percent, the ultrafiltration product with the molecular weight of less than or equal to 3kDa and less than or equal to 10kDa is also higher than that of the primary zymolyte, and the ultrafiltration product with the molecular weight of >10kDa is only slightly lower than that of the ultrafiltration product.

FIG. 3 shows the DPP-IV inhibition of the primary enzyme and three ultrafiltration products from the digestion process of example 3. As can be seen from FIG. 3, the DPP-IV activity inhibition rates of the 4 samples are all obviously reduced, however, the primary zymolyte still reaches 45%, the molecular weight of the primary zymolyte still has nearly 60% of activity, the molecular weight of the primary zymolyte is less than 3kDa and less than or equal to 10kDa, the molecular weight of the primary zymolyte still has nearly 55% of activity, and the molecular weight of the primary zymolyte is more than 10kDa and the molecular weight of the primary zymolyte still has 50% of activity. This shows that the DPP-IV activity inhibition rate is still high although the digestion treatment is carried out, so that the digestion products still have considerable application prospect.

FIG. 4 shows the inhibition of DPP-IV activity of undigested primary enzyme, ultrafiltration product with molecular weight <3kDa, and peptides synthesized in example 4. As can be seen from FIG. 4, the inhibition rate of DPP-IV activity of the synthesized peptide fragment reaches more than 80% compared with more than 60% of the primary zymolyte, 75% of the ultrafiltration product. This shows that the peptide fragment obtained by the screening of example 4 has very high DPP-IV inhibition ability and can be applied to the treatment of type II diabetes.

It should be noted that, in order to reduce the influence of gastrointestinal tract protease on the activity of eggshell membrane hydrolysate or peptide fragment after oral administration, the eggshell membrane hydrolysate or peptide fragment can be loaded and embedded by using a drug delivery carrier commonly used in the market to accelerate the direct absorption of the eggshell membrane hydrolysate or peptide fragment by intestinal tracts. In view of the great demands of oral administration in clinical use today, various new technologies have emerged that are able to overcome the gastrointestinal barrier of peptide fragments, such as enteric coatings, enzyme inhibitors, permeation enhancers, nanoparticles, and intestinal micro-devices. Some new techniques have been developed in clinical trials and even marketed.

For example, in some embodiments, pepsin may cleave a variety of proteins or peptides easily in an acidic environment, but when the pH is >3, pepsin begins to lose its active effect. Therefore, if we can adjust the pH of the microenvironment to 5 or more, it is possible to prevent degradation of the peptide in the stomach.

In some embodiments, an enteric coating is typically used to overcome PP degradation in the stomach. Some organic acids, such as citric acid, are commonly used as pH lowering agents to inhibit intestinal enzyme activity.

Enzyme inhibitors inactivate a target enzyme by reversible or irreversible binding to a specific site of the enzyme, and many chemical molecules can inhibit the activity of the enzyme, such as cholic acid and its derivative diisopropylfluorophosphate. Peptide and modified peptide derived enzyme inhibitors such as aprotinin which inhibits trypsin and chymotrypsin and soybean trypsin which inhibits pancreatic endopeptidase have been widely studied. Chicken and duck egg mucoproteins have recently been developed and are considered safer. They are effective in inhibiting the activity of alpha-chymotrypsin and trypsin and provide 100% protection for insulin. Thus, in some embodiments, this class of enzyme inhibitors may also be used to inhibit the effects of gastrointestinal proteases on the peptide fragments of the present invention.

The colon is a more suitable absorption site than the stomach and small intestine because the enzyme activity at this site is reduced and the pH is neutral. In addition, the residence time at the colon site is longer, with a higher reactivity towards absorption enhancers. The colon thus becomes an ideal site for administration of orally active peptides.

In addition, by encapsulation, the 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 the active peptide from degradation, the particles may also enhance trans-epithelial transport. For example, the encapsulation may be performed in the form of polymer nanoparticles (e.g., polyethylene glycol), lipid nanoparticles (e.g., silica nanoparticles, gold nanoparticles), liposomes, nanoemulsions, inorganic nanoparticles, and the like.

In summary, there are a great number of technical methods available at present, and the eggshell membrane hydrolysate or peptide fragment provided by the present invention can be prepared into products such as medicines or foods which are convenient to take, easy to absorb and have no influence on DPP-IV inhibitory activity.

The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the 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 one 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.