Preparation method and application of narrow gadus bone active peptide compound

Abstract

The invention relates to a preparation method and application of a narrow gadus bone active peptide compound. The preparation method of the narrow gadus bone active peptide compound comprises the following steps: carrying out primary enzymolysis on narrow gadus bone meal by using flavourzyme, separating through ultrafiltration, selecting components with molecular weight less than 3 kDa, and carrying out targeted enzymolysis by using trypsin. Further analyzing and screening the narrow gadus bone active peptide compound to obtain four kinds of narrow gadus bone active peptides, and the amino acid sequences of the four kinds of narrow gadus bone active peptides are FDY, DLGF, DGWR and ECMFPK respectively. The narrow gadus bone active peptide compound and the narrow gadus bone active peptide both have the biological activity of improving insulin resistance and reducing postprandial blood sugar, have the advantages of being good in water solubility, high in safety and the like, and can be applied to the fields of dietary nutrition, biological medicine products and the like.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, and particularly relates to the preparation method and application of the active peptide complex of pollock bone. Background Technology

[0002] Diabetes is mainly classified into three types: gestational diabetes, type 1 diabetes, and type 2 diabetes. Type 2 diabetes mellitus (T2DM) accounts for approximately 90% of all diabetes cases and is a typical chronic metabolic disease, being the most common type of diabetes. Due to changes in diet and physical activity associated with rapid development and urbanization, the age of onset for T2DM is trending younger: the prevalence is 5.0% in the 18-29 age group, 6.5% in the 30-39 age group, and as high as 11.1% in the 40-49 age group. Therefore, the development of drugs for T2DM has become a hot topic.

[0003] Insulin resistance (IR) and β-cell dysfunction are key mechanisms in the development of type 2 diabetes. In Chinese adults, the attributable risk of diabetes due to insulin resistance is twice that of β-cell dysfunction, indicating that insulin resistance is a major driving factor. Currently, research on drugs to combat insulin resistance has increased both domestically and internationally, but it remains in its early stages. Only a small number of health supplements specifically designed to improve insulin resistance are on the market, and their high price and side effects severely limit their application. Therefore, it is necessary to find and screen natural, side-effect-free active substances that can improve IR and hyperglycemia. Among these, bioactive peptides, with their high efficiency and stability, have become highly promising drug candidates in the field of chronic disease treatment. However, the key technology is how to obtain suitable hypoglycemic peptides. Currently, the preparation of bioactive peptides mainly relies on traditional single-enzymatic hydrolysis techniques. However, this method has significant limitations: the enzymatic hydrolysis process is prone to dead zones, is time-consuming, and has uneven effects; at the same time, the product stability is insufficient, the process reproducibility is poor, and the biological activity is generally low. The fundamental reason is that single-enzymatic hydrolysis can usually only effectively cleave the outer amino acid chains of proteins, resulting in low efficiency in the hydrolysis of internal structures.

[0004] As the world's largest aquaculture producer, my country contributes nearly a quarter of the world's total aquatic product output. However, this large-scale export of aquatic products also generates a massive amount of processing waste, including fish skin, scales, swim bladders, and bones. These wastes can account for 50%-70% of the total fish weight, a staggering amount. Among these, Alaska pollock, as one of my country's important fishery resources, generates a huge amount of fish bone waste during processing each year, yet this has not been effectively utilized. The low level of green processing of Alaska pollock byproducts is currently a bottleneck restricting the industry's development. The large-scale disposal of Alaska pollock bones results in enormous economic and resource waste and brings significant environmental pollution pressure.

[0005] Therefore, it is urgent to provide a simple, efficient, and rapid method for preparing active peptides and their complexes from Alaska pollock bone that improve insulin resistance and lower hyperglycemia. Currently, there are no reports on using targeted enzymatic hydrolysis technology on Alaska pollock bone to prepare active peptides and their complexes that lower hyperglycemia and improve insulin resistance. Summary of the Invention

[0006] This invention addresses the issues of low utilization rate and limited activity of pollock bone in existing technologies. It provides a method for preparing pollock bone active peptide complexes that improve insulin resistance and lower blood sugar through targeted enzymatic hydrolysis, as well as the application of pollock bone active peptides.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] The present invention provides a method for preparing a pollock bone active peptide complex, comprising the following steps: preliminary enzymatic hydrolysis of pollock bone using a flavor protease.

[0009] The beneficial effects of adopting the above technical solution include: This invention uses Alaska pollock bone as raw material. Compared with collagen from terrestrial animals, fish bone collagen has advantages such as low immunogenicity (low antigenicity), high bioabsorbability, and a lower risk of disease transmission. Fish bone collagen is rich in hydrophobic amino acids, such as glycine (Gly), proline (Pro), alanine (Ala), and leucine (Leu). These specific amino acids have a positive effect on improving insulin resistance. In the preparation process, this invention uses a flavor protease to initially enzymatically hydrolyze the Alaska pollock bone, releasing its protein components. Compared with other proteases, this invention uses a flavor protease for enzymatic hydrolysis, resulting in better PTP1B inhibitory activity and hypoglycemic activity in the hydrolysate. The above method can obtain an Alaska pollock bone active peptide complex with activities that improve insulin resistance and lower blood sugar. The preparation method provided by this invention has advantages such as high efficiency and good reproducibility, promoting the extension of the Alaska pollock industry chain and improving the degree of deep processing of Alaska pollock industrial by-products. The product prepared by this method can improve insulin resistance and has good hypoglycemic effects.

[0010] Furthermore, the conditions for flavor protease hydrolysis include: pH 7.5, flavor protease addition of 5000 U / g, and hydrolysis at 50℃ for 4 h; and enzyme inactivation after flavor protease hydrolysis.

[0011] Furthermore, the conditions for inactivating the enzyme after flavor protease hydrolysis include: heating in a boiling water bath for 10 minutes.

[0012] Furthermore, the products after enzymatic hydrolysis of flavor protease were ultrafiltered, and components with a molecular weight of <3 kDa were selected.

[0013] The beneficial effects of adopting the above technical solution include: precise separation of enzymatic hydrolysis products through ultrafiltration technology, and effective enrichment of peptides within the target molecular weight range.

[0014] Furthermore, components with a molecular weight <3 kDa were subjected to targeted enzymatic hydrolysis using trypsin.

[0015] The beneficial effects of adopting the above technical solution include: the present invention has found through virtual enzymatic digestion that trypsin digestion can obtain a greater number of polypeptide sequences that conform to the structure-activity relationship of hypoglycemic peptides, thereby making it more advantageous to screen for hypoglycemic peptides.

[0016] Furthermore, the targeted trypsin hydrolysis conditions include: pH 8.0, trypsin addition of 4000 U / g, hydrolysis at 37℃ for 2.5 h; and enzyme inactivation after trypsin hydrolysis.

[0017] Furthermore, the conditions for inactivating the enzyme after targeted trypsin digestion include: heating in a boiling water bath for 5 minutes.

[0018] Furthermore, the process also includes a freeze-drying step to prepare a pollock active peptide complex powder.

[0019] This invention provides a pollock bone bioactive peptide complex, which can be prepared using the method described above. The pollock bone bioactive peptide complex includes one or more of FDY, DLGF, DGWR, and ECMFPK. The pollock bone bioactive peptide complex provided by this invention can improve insulin resistance and lower blood glucose activity. Obtaining the pollock bone bioactive peptide complex from pollock bone is beneficial for increasing the added value of pollock by-products, reducing resource waste, and extending the industrial chain of pollock by-products.

[0020] This invention provides Alaska pollock bone active peptides, wherein the amino acid sequence of the Alaska pollock bone active peptides includes one or more of FDY, DLGF, DGWR, and ECMFPK.

[0021] The beneficial effects of adopting the above technical solution include: the active peptides from the Alaska pollock bone provided by this invention have the effect of improving insulin resistance and lowering blood sugar. Experimental verification shows that the Alaska pollock bone active peptide complex prepared by this invention, along with FDY, DLGF, DGWR, and ECMFPK, can effectively inhibit the activities of PTP1B, DPP-IV, α-glucosidase, and α-amylase. The molecular weights of the Alaska pollock bone active peptides are all less than 1000 Da, belonging to small molecule peptides, which are easily digested and absorbed by the human body, thus facilitating their action on the target site.

[0022] The present invention provides a method for preparing the above-mentioned active peptides of pollock bone, comprising the following steps: solid-phase synthesis of the above-mentioned active peptides of pollock bone.

[0023] The beneficial effects of adopting the above technical solution include: the active peptides of pollock bone prepared by the above method have the effect of improving insulin resistance and lowering blood sugar.

[0024] The present invention provides the use of the above-mentioned pollock bone active peptide complex or the above-mentioned pollock bone active peptide in any one or more of (1) to (6);

[0025] (1) Preparation of hypoglycemic products;

[0026] (2) To prepare products that improve insulin resistance;

[0027] (3) Preparation of PTP1B inhibitors;

[0028] (4) Preparation of DPP-IV inhibitors;

[0029] (5) Preparation of α-glucosidase inhibitors;

[0030] (6) Preparation of α-amylase inhibitors.

[0031] The aforementioned pollock bone active peptide complex or pollock bone active peptide can be prepared by the above method.

[0032] The products mentioned include, but are not limited to, health products, functional foods, special medical foods, dietary supplements, or drugs.

[0033] The beneficial effects of adopting the above technical solution include: the pollock bone active peptide complex and pollock bone active peptide isolated by this invention have a good ability to improve insulin resistance, have excellent hypoglycemic effects, can reduce postprandial blood glucose, have high application development value, and can be used as functional ingredients in health products, medicines and other fields. Specifically, the PTP1B inhibition rate of the pollock bone active peptide complex is 84.38±0.60%, the DPP-IV inhibition rate is 36.07±1.18%, the α-glucosidase inhibition rate is 29.27±1.04%, and the α-amylase inhibition rate is 24.53±0.84%. The glucose consumption of the pollock bone active peptide complex (10.06 mM) is 44.75% higher than that of the model group (6.95 mM). FDY, DLGF, DGWR, and ECMFPK can all effectively inhibit the activities of PTP1B, DPP-IV, α-glucosidase and α-amylase, and their PTP1B inhibition rate IC50 is significantly higher than that of the model group. 50 The concentrations were 0.8291 mg / mL, 1.247 mg / mL, 0.03766 mg / mL, and 0.3345 mg / mL, respectively; the IC50 of DPP-IV inhibition was [missing value]. 50The concentrations were 1.187 mg / mL, 0.8934 mg / mL, 1.429 mg / mL, and 0.582 mg / mL, respectively; the IC50 of its α-glucosidase inhibition was... 50 The IC50 values ​​were 1.153 mg / mL, 0.9524 mg / mL, 0.8182 mg / mL, and 1.271 mg / mL, respectively, and the IC50 values ​​for α-amylase inhibition rate were also [not specified]. 50 The concentrations were 1.363 mg / mL, 0.7495 mg / mL, 1.209 mg / mL, and 1.613 mg / mL, respectively. Attached Figure Description

[0034] Figure 1 The results of Example 4 are shown below. The horizontal axis represents the types of active peptide complexes in Alaska pollock bone, and the vertical axis represents the inhibition rate. Among them, A represents the effect of active peptide complexes in Alaska pollock bone (FP3 and FP3-Try) on the inhibition rate of PTP1B; B represents the effect of active peptide complexes in Alaska pollock bone (FP3 and FP3-Try) on the inhibition rate of DPP-IV; C represents the effect of active peptide complexes in Alaska pollock bone (FP3 and FP3-Try) on the inhibition rate of α-glucosidase; and D represents the effect of active peptide complexes in Alaska pollock bone (FP3 and FP3-Try) on the inhibition rate of α-amylase.

[0035] Figure 2 The effects of different concentrations of FP3-Try on the survival rate of HepG2 cells (A) and the effects of different concentrations of FP3-Try on glucose consumption of IR-HepG2 cells (B) are shown. In A, the x-axis represents the concentration of the pollock bone active peptide complex (FP3-Try), and the y-axis represents its effect on the survival rate of HepG2 cells. In B, the x-axis represents the concentration of the pollock bone active peptide complex (FP3-Try), and the y-axis represents the glucose consumption of the cells.

[0036] Figure 3 The results show the molecular docking of the active peptide FDY from Alaska pollock bone. From top to bottom, these are the docking results of FDY with the target sites PTP1B, DPP-IV, α-glucosidase, and α-amylase, respectively.

[0037] Figure 4 The results show the molecular docking of the active peptide DLGF from Alaska pollock bone. From top to bottom, the results show the docking of DLGF with the target sites PTP1B, DPP-IV, α-glucosidase, and α-amylase, respectively.

[0038] Figure 5 The results show the molecular docking of the active peptide DGWR from Alaska pollock bone. From top to bottom, these are the docking results of DGWR with the target sites PTP1B, DPP-IV, α-glucosidase, and α-amylase.

[0039] Figure 6 The results show the molecular docking of ECMFPK, an active peptide from Alaska pollock bone. From top to bottom, these are the docking results of ECMFPK with the target sites PTP1B, DPP-IV, and α-glucosidase.

[0040] Figure 7 The effects of FDY, DLGF, DGWR, and ECMFPK on the inhibition rates of PTP1B (A), DPP-IV (B), α-glucosidase (C), and α-amylase (D) are shown. The x-axis represents the concentration of active peptides, and the y-axis represents the inhibition rate. Detailed Implementation

[0041] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0042] This invention provides a method for preparing a pollock bone bioactive peptide complex and its application. The method for preparing the pollock bone bioactive peptide complex includes the following steps: Pollock bone is initially enzymatically hydrolyzed using a flavoring enzyme, separated by ultrafiltration to obtain components with a molecular weight <3 kDa; the peptide sequences of the components with a molecular weight <3 kDa are analyzed using liquid chromatography-mass spectrometry; computer-aided enzymatic hydrolysis technology is used to virtually screen the components with a molecular weight <3 kDa; the trypsin selected by the computer is used to target and hydrolyze the components with a molecular weight <3 kDa; and the target pollock bone bioactive peptide complex is obtained through in vitro verification. Finally, pollock bone bioactive peptides are obtained from the targeted enzymatic hydrolysis products by combining bioinformatics, molecular docking technology, and in vitro verification. The amino acid sequence of the pollock bone bioactive peptide includes one or more of FDY, DLGF, DGWR, and ECMFPK. This invention utilizes computer-aided targeted enzymatic hydrolysis technology to achieve breakthrough optimization through three levels of precise regulation: Based on a <3kDa peptide sequence database resolved by LC-MS, combined with bioinformatics simulation of optimal enzyme cleavage sites, the invention completely avoids the enzymatic hydrolysis dead zones and random cleavage problems of traditional methods; by directionally enriching functional peptides containing hypoglycemic hydrophobic amino acids (Gly / Pro / Ala / Leu), the product possesses activity in improving insulin resistance, with a target peptide yield increase of >40%; simultaneously, the hydrophilic design of the peptide chain is optimized to reduce the exposure of hydrophobic residues, resulting in a 2-fold increase in product water solubility and a significant improvement in safety. This provides an innovative path for the precise industrial preparation of highly active hypoglycemic peptides. The Alaska pollock bone active peptide complex and Alaska pollock bone active peptide provided by this invention both possess bioactivity in improving insulin resistance and reducing postprandial blood glucose, and also have the advantages of good water solubility and high safety, making them applicable to fields such as dietary nutrition and biopharmaceutical products.

[0043] In the above method, the pollock bone can be freeze-dried pollock bone powder.

[0044] The preliminary enzymatic hydrolysis of Alaska pollock bones using a flavor protease can include the following steps: using Alaska pollock bones as raw material, preliminary enzymatic hydrolysis is performed using a flavor protease with an enzyme activity to substrate mass ratio of 5000 U / g, a hydrolysis time of 4 hours, a hydrolysis temperature of 50℃, and a pH of 7.5. After hydrolysis, the mixture is heated in a boiling water bath for 10 minutes to inactivate the enzyme, centrifuged at 10000 r / min for 20 minutes, and the supernatant is collected to obtain the Alaska pollock bone hydrolysate.

[0045] The separation of active peptide complexes from pollock bone can include the following steps: fractionating the pollock bone enzymatic hydrolysate through 5 kDa and 3 kDa ultrafiltration membranes, and freeze-drying the fractions with a concentration <3 kDa.

[0046] The <3 kDa component was further targeted by trypsin enzymatic hydrolysis, including the following steps: the ratio of trypsin activity to substrate mass was 4000 U / g, the hydrolysis time was 2.5 h, the hydrolysis temperature was 37℃, and the pH was 8.0. Then, the enzyme was inactivated by heating in a boiling water bath for 5 min, followed by freeze drying to obtain a composite powder of pollock bone active peptides with high hypoglycemic activity and high improvement of insulin resistance.

[0047] This invention provides a pollock bone active peptide complex, which can be prepared using the method described above. Experimental verification shows that this pollock bone active peptide complex contains one or more of FDY, DLGF, DGWR, and ECMFP.

[0048] The present invention also provides the application of the above-mentioned pollock active peptide complex in the preparation of products with hypoglycemic or insulin resistance-improving activity.

[0049] This invention addresses the low utilization rate and limited activity of existing pollock bone methods by providing a method for obtaining pollock bone bioactive peptide complexes based on initial enzymatic hydrolysis with flavor protease, separation and purification, computer-aided virtual enzymatic screening, and targeted trypsin hydrolysis. This invention utilizes ultrafiltration separation technology, protein databases and online software, and employs multiple screening methods based on properties such as PTP1B, DPP-IV, α-glucosidase, and α-amylase inhibitory activities. Results show that the pollock bone bioactive peptide complexes obtained by this invention possess advantages such as good water solubility, good safety, excellent PTP1B inhibition ability, and the ability to improve insulin resistance and reduce hyperglycemia.

[0050] This invention also provides Alaska pollock bone bioactive peptides that lower blood sugar and improve insulin resistance, their preparation method, and their applications.

[0051] This invention provides four active peptides from Alaska pollock bone that lower blood sugar and improve insulin resistance, with amino acid sequences of FDY, DLGF, DGWR, and ECMFP, respectively. These active peptides from Alaska pollock bone can improve insulin resistance and lower blood sugar.

[0052] The preparation method of the above-mentioned active peptides of Alaska pollock bone may include the following steps: solid-phase synthesis of active peptides of Alaska pollock bone having the above-mentioned amino acid sequence.

[0053] This invention provides a method for screening the above-mentioned active peptides from pollock bone, comprising the following steps:

[0054] (a) The peptide amino acid sequence of the complex obtained by the above-mentioned targeted enzymatic hydrolysis that improves insulin resistance and lowers blood glucose was identified by liquid chromatography-mass spectrometry.

[0055] (b) Using computer-aided screening technology, peptides with known amino acid sequences obtained in step (a) are screened to obtain peptides with potential high activity in improving insulin resistance and lowering blood sugar.

[0056] (c) Screen peptides by solid-phase synthesis and in vitro verification of their inhibitory activities on PTP1B, DPP-IV, α-glucosidase and α-amylase.

[0057] This invention employs liquid chromatography-mass spectrometry (LC-MS) to deeply analyze the fine composition and sequence information of <3 kDa peptides obtained by ultrafiltration. Based on this, it innovatively utilizes computer-aided enzymatic digestion technology to optimize targeted enzymatic digestion strategies for the ultrafiltration components based on the obtained peptide composition data. It simulates and predicts optimal digestion conditions and enzyme types, aiming to directionally release or enrich peptide sequences with specific biological activities. Finally, through in vitro functional validation, it successfully screened and obtained active peptides from Alaska pollock bone that improve insulin resistance and lower blood sugar.

[0058] In step (a) above, the identification of active peptides from Alaska pollock bone may include the following steps: Amino acid sequence identification of the components obtained from targeted enzymatic digestion using liquid chromatography-mass spectrometry (LC-MS / MS). Mobile phase A is an acetonitrile-formic acid aqueous solution with a volume ratio of acetonitrile, formic acid, and water of 2:0.1:97.9; mobile phase B is an acetonitrile solution of formic acid with a volume fraction of 0.1% formic acid, using acetonitrile as the solvent; the gradient starts at 2% (volume percentage) of phase B, increases to 35% (volume percentage) in a non-linear gradient over 47 min, then increases to 100% (volume percentage) within 1 min and is maintained for 12 min; the loading volume is 1.5 μL, and the column flow rate is 300 nL / min.

[0059] In step (b) above, screening for bioactive peptides from Alaska pollock bone may include the following steps: Preliminary screening of bioactive peptides identified by LC-MS / MS based on peptide chain length and hydrophobicity; prediction of peptide bioactivity values ​​using the online tool PeptideRanker; prediction of peptide water solubility and toxicity using the online tools Innovagen and ToxinPred; and molecular docking of bioactive peptide sequences that are both water-soluble and non-toxic.

[0060] This invention utilizes protein databases and online software, and employs multiple screening methods based on properties such as PTP1B, DPP-IV, α-glucosidase, α-amylase inhibitory activity, water solubility, and biotoxicity. Molecular docking was used to explore the interaction mechanisms between the active peptides and receptors PTP1B (1Q6J), DPP-IV (5J3J), α-glucosidase (2QMJ), and α-amylase (1PIF). Finally, solid-phase synthesis was used to verify the peptides' activity in improving insulin resistance and lowering blood glucose. Results show that the four active peptides screened in this invention all possess advantages such as good water solubility, good safety, excellent inhibition of PTP1B, DPP-IV, α-glucosidase, and α-amylase, and the ability to lower blood glucose and improve insulin resistance.

[0061] In this example, pollock bone powder was purchased from Shandong Rongcheng Taixiang Food Co., Ltd.; flavor protease, papain, neutral protease, alkaline protease, trypsin, pepsin, PBS buffer, Tris-HCl buffer, HEPES buffer, p-nitrophenyl-α-D-glucopyranoside (pNPP), N-glycylprolyl-p-nitroaniline hydrochloride (Gly-Pro-pNA), and soluble starch were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; PTP1B enzyme and DPP-IV enzyme were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; α-amylase and α-glucosidase were purchased from Hefei Bomei Biotechnology Co., Ltd.; DMEM complete culture medium, fetal bovine serum, and HepG-2 cells were all purchased from Wuhan Pronosei Life Science Co., Ltd.; metformin hydrochloride, insulin resistance agent (glucosamine), penicillin-streptomycin solution (100X), and CCK-8 kit were all purchased from Beyotime Biotechnology Co., Ltd. 3,5-Dinitrosalicylic acid (DNS) chromogenic reagent was purchased from Guangzhou Hewei Pharmaceutical Technology Co., Ltd.; glucose (Glu) test kit was purchased from Nanjing Jiancheng Bioengineering Institute.

[0062] Unless otherwise specified, all techniques or conditions used in the embodiments are conventional methods or performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels or prepared according to conventional methods in this field. Instruments used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels. Unless otherwise specified, the solvent for all solutions involved in this invention is water.

[0063] The following is a description through specific embodiments.

[0064] Example 1

[0065] Pollock bones were dried and pulverized to obtain pollock bone powder. The pollock bone powder was mixed with deionized water at a ratio of 1 g:30 mL. Different proteases were added, each at a concentration of 5000 U / g (5000 U of protease per gram of pollock bone powder). The proteases included alkaline protease, flavor protease, papain, neutral protease, trypsin, and pepsin. The optimal pH for alkaline protease was 10.5, and the optimal temperature was 45℃. The optimal pH for flavor protease was 7.5, and the optimal temperature was 50℃. The optimal pH for papain was 6, and the optimal temperature was 55℃. The optimal pH for neutral protease was 7, and the optimal temperature was 50℃. The optimal pH for trypsin was 7.5, and the optimal temperature was 37℃. The optimal pH for pepsin was 3, and the optimal temperature was 37℃. Hydrolysis was carried out for 4 hours at the optimal pH and temperature for each enzyme, with continuous stirring at 400 rpm during the process. Subsequently, the enzymatic hydrolysis reaction was terminated by boiling in a water bath for 10 min, centrifuged at 10000 r / min for 20 min, and the supernatant was collected as the enzymatic hydrolysate of Alaska pollock bone. The hydrolysate was then freeze-dried to obtain the active peptide complex of Alaska pollock bone for subsequent analysis.

[0066] Using the active peptide complex of Alaska pollock bone as the test sample, the ability of the active peptide complex of Alaska pollock bone to improve insulin resistance (PTP1B inhibition activity) and its hypoglycemic activity (DPP-IV, α-glucosidase and α-amylase inhibition activities) were detected.

[0067] (1) The PTP1B inhibition rate was detected, including the following steps: setting up a sample group, a sample control group, and a blank group. The reaction solution groups and amounts are shown in Table 1. The test sample solution, PTP1B enzyme solution, and pNPP solution were all prepared using HEPES buffer (pH=7.4) as the solvent.

[0068] Table 1. Grouping and Dosage of Reaction Solution

[0069]

[0070] According to Table 1, add HEPES buffer, the sample solution to be tested, and PTP1B enzyme solution, mix well, and incubate at 37°C in the dark for 10 min. Then add 50 μL of pNPP solution (pNPP concentration of 5 mM) to the system, and continue incubation at 37°C in the dark for 24 h. Measure the absorbance of the solution at a wavelength of 405 nm.

[0071] The PTP1B inhibitory activity (i.e., the PTP1B inhibition rate) is calculated using the following formula.

[0072] ;

[0073] In the formula, A 样品组 A represents the absorbance of the sample group at 405 nm. 样品对照组 The absorbance of the sample control group at 405 nm; A 空白组 The absorbance of the blank group at 405 nm is given.

[0074] (2) The DPP-IV inhibition rate was detected, including the following steps: setting up a sample group, a sample control group, a blank group, and a blank control group. The reaction solution groups and amounts are shown in Table 2. The test sample solution, DPP-IV enzyme solution, and Gly-Pro-pNA were all prepared using Tris-HCl buffer (pH=8.0) as the solvent.

[0075] Table 2. Grouping and Dosage of Reaction Solution

[0076]

[0077] According to Table 2, add Tris-HCl buffer (pH=8.0), the sample solution to be tested, and DPP-IV enzyme solution, mix well, and incubate at 37℃ for 10 min. Add 25 μL of Gly-Pro-p-nitroanilide hydrochloride solution (Gly-Pro-pNA, 1.6 mM), and continue incubation at 37℃ for 1 h. Measure the absorbance of the solution at 405 nm. Calculate the DPP-IV inhibitory activity (i.e., DPP-IV inhibition rate) using the following formula.

[0078] ;

[0079] In the formula, A 样品组 A represents the absorbance value of the sample group at 405 nm. 样品对照组 The absorbance value of the control group at 405 nm; A 空白组 The absorbance of the blank group was measured at 405 nm; A 空白对照组 The absorbance value of the blank group was measured at 405 nm.

[0080] (3) The α-glucosidase inhibition rate was detected, including the following steps: setting up a sample group, a sample control group, and a blank group. The reaction solution groups and amounts are shown in Table 3. The Na2CO3 solution was prepared with deionized water, and the test sample solution, α-glucosidase solution, and pNPP solution were all prepared with PBS buffer (pH=6.8) as the solvent.

[0081] Table 3. Grouping and Dosage of Reaction Solution

[0082]

[0083] According to Table 3, mix the PBS buffer, the sample solution, and the α-glucosidase solution thoroughly and incubate at 37°C for 10 min. Then add 25 μL of pNPP solution (5 mM pNPP concentration) to the system and continue incubation at 37°C for 15 min. Finally, add 75 μL of Na2CO3 solution (1 M Na2CO3 concentration) to terminate the reaction and measure the absorbance of the solution at 405 nm. Calculate the α-glucosidase inhibitory activity (i.e., α-glucosidase inhibition rate) using the following formula.

[0084] ;

[0085] In the formula, A 样品组 A represents the absorbance of the sample group at 405 nm. 样品对照组 The absorbance of the sample control group at 405 nm; A 空白组 The absorbance of the blank group at 405 nm is given.

[0086] (4) Detection of α-amylase inhibition rate, including the following steps: setting up sample group, control group and blank group. The reaction solution grouping and dosage are shown in Table 4. The test sample solution, α-amylase solution and soluble starch solution were all prepared with PBS buffer (pH=6.8) as solvent.

[0087] Table 4. Grouping and Dosage of Reaction Solution

[0088]

[0089] According to Table 4, mix PBS buffer (pH=6.8), α-amylase solution (0.1 U / mL), and the sample solution to be tested thoroughly, and incubate at 37°C for 10 min. Then add 100 μL of soluble starch solution to the system, continue incubating at 37°C for 5 min, add 100 μL of 3,5-dinitrosalicylic acid (DNS), incubate in a water bath at 100°C for 10 min, and finally dilute with 4 mL of deionized water. Measure the absorbance of the solution at 405 nm. Calculate the α-amylase inhibitory activity (i.e., α-amylase inhibition rate) using the following formula.

[0090] ;

[0091] In the formula, A 样品组 A represents the absorbance of the sample group at 405 nm. 样品对照组 The absorbance of the sample control group at 405 nm; A 空白组 The absorbance of the blank group at 405 nm is given.

[0092] The results of PTP1B inhibitory activity and hypoglycemic activity (DPP-IV, α-glucosidase, α-amylase inhibitory activity) detection are shown in Tables 5 and 6. The highest inhibition rate was scored as 6 points, with scores decreasing sequentially according to inhibition rate. The Alaska pollock bone active peptide complex treated with flavor protease had a total score of 22 points, and flavor protease was selected as the initial enzymatic digestion protease. It can be seen that the Alaska pollock bone active peptide complex prepared with flavor protease exhibits superior overall PTP1B inhibitory activity and hypoglycemic activity (DPP-IV, α-glucosidase, α-amylase inhibitory activity) compared to samples prepared with other enzymes.

[0093] Table 5. Bioactivity evaluation of the active peptide complexes of Alaska pollock bone obtained by enzymatic hydrolysis with different proteases.

[0094]

[0095] Table 6. Bioactivity scores of Alaska pollock bone bioactive peptide complexes obtained by enzymatic hydrolysis with different proteases

[0096]

[0097] Example 2

[0098] The flavor protease hydrolysate (FP) obtained from the flavor protease hydrolysis of Alaska pollock bones in Example 1 was sequentially passed through ultrafiltration membranes with molecular weights of 5 kDa and 3 kDa. Fractions with molecular weights >5 kDa (FP5), 3 kDa-5 kDa fractions (FP3-5), and <3 kDa fractions (FP3) were collected. The ultrafiltration fractions were lyophilized, and the PTP1B inhibitory activity, DPP-IV inhibitory activity, α-glucosidase inhibitory activity, and α-amylase inhibitory activity of each collected fraction were determined using the same methods as in Example 1.

[0099] Based on the screening results, the components with higher inhibition rates were selected and freeze-dried for later use. The screening results are shown in Table 7. It can be seen that the PTP1B inhibitory activity, DPP-IV inhibitory activity, α-glucosidase inhibitory activity, and α-amylase inhibitory activity of the component with a molecular weight <3 kDa (FP3) are significantly better than those of other components. Therefore, the component with a molecular weight <3 kDa (FP3) was selected for subsequent targeted enzymatic hydrolysis experiments.

[0100] Table 7 Bioactivity of different target sites of ultrafiltration components

[0101]

[0102] Example 3

[0103] The amino acid sequence of the <3 kDa fraction (FP3) of the pollock bone active peptide complex was identified by Beijing Baitaipaike Biotechnology Co., Ltd., including the following steps: Liquid chromatography-mass spectrometry (LS-MS / MS) was used to perform secondary mass spectrometry analysis on the <3 kDa fraction. Mobile phase A was an acetonitrile-formic acid aqueous solution with a volume ratio of acetonitrile, formic acid, and water of 2:0.1:97.9; mobile phase B was a formic acid solution in acetonitrile with a formic acid volume fraction of 0.1% and acetonitrile as the solvent; the gradient started at 2% of phase B, increased to 35% nonlinearly over 47 min, then increased to 100% within 1 min and maintained for 12 min, all of which are volume percentages; the sample loading volume was 1.5 μL, and the column flow rate was 300 nL / min.

[0104] By comparing with protein databases, the amino acid sequences of 670 novel bioactive peptides were obtained. Based on the structure-activity relationship of hypoglycemic peptides, the N-terminus of these peptides is predominantly composed of leucine (Leu), glycine (Gly), phenylalanine (Phe), methionine (Met), or isoleucine (Ile), while the C-terminus is predominantly composed of arginine (Arg), proline (Pro), phenylalanine (Phe), leucine (Leu), or lysine (Lys). Among these 670 novel bioactive peptide amino acid sequences, 62 peptide sequences conform to the structure-activity relationship of hypoglycemic peptides.

[0105] The ANALYSIS-ENZYME(S):ACTION tool in the online tool BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) was used to perform virtual enzymatic digestion on 670 polypeptide sequences in the <3 kDa fraction (FP3). The proteases used for virtual digestion included trypsin, papain, proteinase K, Bacillus subtilis protease, chymotrypsin A, chymotrypsin C, and pepsin (pH > 2). The proportion of polypeptide sequences with N-terminal amino acids of L / G / F / M / I and C-terminal amino acids of R / P / F / L / K was analyzed after virtual digestion. Proteases for subsequent targeted digestion were screened based on the N / C-terminal proportions.

[0106] The results of the virtual enzymatic digestion are shown in Table 8. After trypsin digestion, the number of peptides with corresponding N-terminal amino acids increased by 12.93% compared to the <3 kDa component (FP3); the number of peptides with corresponding C-terminal amino acids increased by 19.48% compared to the <3 kDa (FP3) component. Among these, 75 peptide sequences conforming to the structure-activity relationship of hypoglycemic peptides were identified after virtual trypsin digestion, an increase of 13 sequences compared to the <3 kDa component. Therefore, trypsin was chosen for the subsequent targeted digestion of the <3 kDa component (FP3).

[0107] Table 8. Hypoglycemic active sites after virtual enzymatic hydrolysis

[0108]

[0109] Note: N-terminus (%) refers to the percentage of peptides with the corresponding N-terminal amino acid (L / G / F / M / I) out of the total number of peptides. C-terminus (%) refers to the percentage of peptides with the corresponding C-terminal amino acid (R / P / F / L / K) out of the total number of peptides.

[0110] Example 4

[0111] The fraction with a molecular weight <3 kDa (FP3) was targeted enzymatically hydrolyzed using trypsin, including the following steps: The lyophilized powder of the fraction with a molecular weight <3 kDa (FP3) was dissolved in deionized water at a mass-to-volume ratio of 1 g:30 mL. Trypsin (4000 U / g), i.e., 4000 U of trypsin per gram of FP3 lyophilized powder, was added. Hydrolysis was carried out at the optimal pH of 8.0 and the optimal temperature of 37°C for 2.5 h, with continuous stirring at 200 rpm during the process. Subsequently, the enzymatic hydrolysis was terminated by boiling in a water bath for 5 min, followed by freeze-drying to obtain the Alaska pollock bone active peptide complex FP3-Try, which was used for subsequent analysis to evaluate the inhibitory activities of PTP1B, DPP-IV, α-glucosidase, and α-amylase. The detection methods were as described in Example 1.

[0112] Test results as follows Figure 1As shown, the Alaska pollock bone bioactive peptide complex FP3-Try, after trypsin-targeted hydrolysis, exhibits excellent inhibitory activity against PTP1B, DPP-IV, α-glucosidase, and α-amylase. The PTP1B inhibitory activity increased from 72.90% to 84.38%, a 15.75% increase compared to the <3 kDa fraction; the DPP-IV inhibitory activity increased from 23.30% to 36.07%, a 54.81% increase compared to the <3 kDa fraction; the α-glucosidase inhibitory activity increased from 25.97% to 29.27%, a 12.71% increase compared to the <3 kDa fraction; and the α-amylase inhibitory activity increased from 19.49% to 24.53%, a 25.86% increase compared to the <3 kDa fraction. These results indicate that trypsin-targeted hydrolysis significantly enhances the inhibitory activities of PTP1B, DPP-IV, α-glucosidase, and α-amylase.

[0113] Example 5

[0114] The amino acid sequence of the pollock bone active peptide complex FP3-Try was identified by Beijing Baitaipaike Biotechnology Co., Ltd., including the following steps: FP3-Try was analyzed by secondary mass spectrometry using liquid chromatography-mass spectrometry (LS-MS / MS). Mobile phase A was an acetonitrile-formic acid aqueous solution with a volume ratio of acetonitrile, formic acid and water of 2:0.1:97.9; mobile phase B was a formic acid solution in acetonitrile with a volume fraction of 0.1% formic acid, and the solvent was acetonitrile; the gradient started from 2% of phase B, increased to 35% of phase B in a non-linear gradient over 47 min, and then increased to 100% of phase B within 1 min, and maintained for 12 min. All percentages are volume percentages; the sample loading volume was 1.5 μL, and the column flow rate was 300 nL / min.

[0115] By comparing with protein databases, amino acid sequences of 537 bioactive peptides were obtained, of which 107 peptide sequences conformed to the structure-activity relationship of hypoglycemic peptides. This is an increase of 45 peptide sequences conforming to the structure-activity relationship of hypoglycemic peptides compared with the molecular weight <3 kDa component (FP3), and an increase of 32 peptide sequences conforming to the structure-activity relationship of hypoglycemic peptides compared with the number of peptide sequences conforming to the structure-activity relationship of hypoglycemic peptides after virtual digestion with trypsin.

[0116] As shown in Table 9, after targeted hydrolysis with trypsin, the number of peptides corresponding to the N-terminal amino acids in FP3-Try increased by 49.64% compared with the molecular weight <3 kDa component (FP3); the number of peptides corresponding to the C-terminal amino acids in FP3-Try increased by 30.31% compared with the molecular weight <3 kDa component (FP3). These results indicate that targeted hydrolysis with trypsin can enrich hypoglycemic peptides.

[0117] Table 9. Hypoglycemic active sites after trypsin targeted enzymatic hydrolysis

[0118]

[0119] Note: N-terminus (%) refers to the percentage of peptides with the corresponding N-terminal amino acid (L / G / F / M / I) out of the total number of peptides. C-terminus (%) refers to the percentage of peptides with the corresponding C-terminal amino acid (R / P / F / L / K) out of the total number of peptides.

[0120] Example 6

[0121] The biocompatibility of the Alaska pollock bone bioactive peptide complex FP3-Try was evaluated using the CCK-8 assay. The experimental method included the following steps:

[0122] (1) Set up a blank group. Add 100 μL of DMEM complete culture medium without HepG2 cells to the wells of the 96-well plate.

[0123] (2) HepG2 cells were cultured in DMEM complete medium until the logarithmic growth phase. 100 μL of DMEM complete medium containing HepG2 cells was added to each well of a 96-well plate, resulting in a HepG2 cell density of 1 × 10⁻⁶ cells / well. 4 Cells / well, 6 replicates. After culturing for 24 h until HepG2 cells adhered, the culture medium was aspirated. Cells were divided into control and experimental groups. In the experimental group, 100 μL of FP3-Try solution at different concentrations (0 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, and 8 mg / mL, dissolved in complete DMEM medium) was added to the wells containing HepG2 cells. In the control group, 100 μL of DMEM medium without peptide samples was added to the wells containing HepG2 cells.

[0124] (3) Add 10 μL of CCK-8 solution (Beyotime Biotechnology Co., Ltd., C0037) to each well of each group, incubate at 37℃ for 1 h, measure the absorbance at 450 nm, and calculate the cell viability according to the following formula.

[0125] ;

[0126] Experimental results are as follows Figure 2 As shown in Figure A, different concentrations of FP3-Try had no effect on the survival rate of HepG-2 cells, indicating that the polypeptide complex prepared in this invention has no toxic side effects.

[0127] Example 7

[0128] The experimental method for detecting cellular glucose consumption of the Alaska pollock bone bioactive peptide complex FP3-Try includes the following steps:

[0129] (1) Set up a control group (C), an experimental group, a model group (M), and a positive drug group (Met), and set up 3 parallel groups.

[0130] Experimental group: HepG-2 human liver cancer cells were cultured in DMEM complete medium containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin solution (100X) and cultured at 37°C in a 5% CO2 cell culture incubator. HepG-2 cells in the logarithmic growth phase were seeded into 6-well plates at a cell density of 1×10⁻⁶ cells / well. 6 / well, incubated at 37℃ for 24 h. Add 2.5 mL of FP3-Try solution of different concentrations (FP3-Try concentrations of 0.25 mg / mL, 1 mg / mL, and 2 mg / mL, respectively). The FP3-Try solution is prepared with 16 mM glucosamine solution. The preparation method of 16 mM glucosamine solution is as follows: weigh glucosamine, dissolve it in DMEM complete medium, vortex mix well, and prepare a 16 mM glucosamine solution (i.e., modeling reagent).

[0131] Control group: The control group was treated the same as the experimental group except that the FP3-Try solution was replaced with DMEM complete culture medium.

[0132] Model group: Contains no FP3-Try. The model group is prepared using a 16 mM glucosamine solution instead of the FP3-Try solution, following the same preparation method as the experimental group. Everything else is the same as the experimental group.

[0133] Positive drug group: Metformin hydrochloride was used as the positive drug. In addition to the experimental group, 2.5 mL of 200 μM metformin hydrochloride solution was used instead of FP3-Try solution. The metformin hydrochloride solution was prepared with 16 mM glucosamine solution in the same way as above. The rest was the same as the experimental group.

[0134] (3) After culturing each group at 37℃ and 5% CO2 for 24 h, the culture medium was removed and used as the test samples. The glucose content was determined using a glucose (Glu) kit purchased from Beyotime. A new 96-well plate was used, and 200 μL of glucose assay working solution was added to each well. Sample group: After adding 200 μL of glucose assay working solution to each well, 2 μL of the sample to be tested was added to each well, mixed well, and incubated at 37℃ for 10 min. Blank group: After adding 200 μL of glucose assay working solution to each well, 2 μL of ultrapure water was added to each well, mixed well, and incubated at 37℃ for 10 min. Calibration group: After adding 200 μL of glucose assay working solution to each well, 2 μL of calibrator was added to each well, mixed well, and incubated at 37℃ for 10 min. The absorbance value at 505 nm was measured, and the glucose content (i.e., the remaining glucose content) and glucose consumption in the culture medium of each group of cells after 24 h of culture were calculated based on the absorbance value. The calculation formula is: Residual glucose content (mM) = (OD) 样品组 -OD 空白组 ) / (OD 校准组 -OD 空白组 × Calibration solution concentration.

[0135] Glucose consumption (mM) = Glucose content in DMEM complete medium - Remaining glucose content

[0136] Experimental results are as follows Figure 2 As shown in Figure B, FP3-Try significantly increased cellular glucose consumption in insulin-resistant states in a concentration-dependent manner. Compared with the model group (6.95±0.32 mM), glucose consumption in HepG2 cells gradually increased with increasing FP3-Try concentration. Specifically, 2 mg / mL FP3-Try increased cellular glucose consumption to 10.06±0.21 mM, an increase of approximately 44.75% compared to the model group.

[0137] Example 8

[0138] Screening of Alaska pollock bone bioactive peptides: Based on the amino acid sequence identification results of the FP3-Try complex of Alaska pollock bone bioactive peptides in Example 5, peptide sequences were initially screened according to molecular weight and amino acid number. Peptides with a molecular weight less than 1000 Da and ≤6 amino acids were selected for further screening. The bioactivity values ​​of the peptides were predicted using the online tool PeptideRanker (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.php), and peptides with predicted bioactivity values ​​greater than 0.8 were retained. The water solubility and biotoxicity of the peptides were predicted using the online tools Innovagen (www.innovagen.com / proteomicstools) and ToxinPred (http: / / crdd.osdd.net / raghava / / toxinpred), and peptides showing "good solubility" and non-toxicity were retained. Twenty-nine bioactive peptides were screened using the above methods. The predicted bioactivity, toxicity, solubility, and other characteristics of FDY, DLGF, DGWR, and ECMFPK are shown in Table 10.

[0139] Table 10. Predicted bioactivity, toxicity, solubility, and other properties of bioactive peptides from Alaska pollock bone.

[0140]

[0141] Molecular construction and binding energy optimization were performed on the 29 peptide fragments mentioned above using Discovery Studio software. The high-throughput screening program LibDock in Discovery Studio was selected, with peptide molecules as ligands and optimized target proteins PTP1B (ID: 1Q6J), DPP-IV (ID: 5J3J), α-glucosidase (ID: 2QMJ), and α-amylase (ID: 1PIF) from the PDB library as acceptors for preliminary molecular docking. The docking results with the highest LibDock scores were further optimized using the semi-flexible precision screening program CDOCKER with its built-in CHARMM force field. The optimized results were screened according to the -CDOCKER ENERGY value, finally yielding four bioactive peptides with amino acid sequences of FDY, DLGF, DGWR, and ECMFPK. These bioactive peptides exhibit advantages such as good biological activity, good water solubility, and non-toxicity.

[0142] The 3D and 2D interaction diagrams of peptides FDY, DLGF, DGWR, ECMFPK with multiple targets are shown below. Figures 3 to 6As shown in Tables 11 to 14, these tables describe the detailed docking results of the four peptides with their targets (PTP1B / DPP-IV / α-glucosidase / α-amylase). It can be seen that the four peptides exhibited the best binding affinity and highest stability to the multiple targets (PTP1B / DPP-IV / α-glucosidase / α-amylase). The high binding energy indicates a strong affinity between the receptor and ligand. The peptides formed favorable bindings to PTP1B / DPP-IV / α-glucosidase / α-amylase through hydrogen bonds, salt bridges, hydrophobic interactions, and van der Waals forces.

[0143] Table 11. Docking results of peptide FDY with multiple targets

[0144]

[0145] Table 12. Docking results of peptide DLGF with multiple targets

[0146]

[0147] Note: "-" in the table indicates that there is no corresponding force.

[0148] Table 13. Docking results of peptide DGWR with multiple targets

[0149]

[0150] Note: "-" in the table indicates that there is no corresponding force.

[0151] Table 14. Docking results of peptide ECMFPK with multiple targets

[0152]

[0153] Note: "-" in the table indicates that there is no corresponding force.

[0154] Example 9

[0155] In vitro activity validation of bioactive peptides FDY, DLGF, DGWR, and ECMFPK: Four bioactive peptides were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) using a chemical solid-phase synthesis method. The amino acid sequences of the bioactive peptides were FDY, DLGF, DGWR, and ECMFPK, respectively, with a purity exceeding 98%. Subsequently, the in vitro inhibitory activities of the above bioactive peptides against PTP1B, DPP-IV, α-glucosidase, and α-amylase were validated.

[0156] In the PTP1B inhibition assay, the concentrations of the active peptide were set to 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively. In the DPP-IV, α-glucosidase, and α-amylase inhibition assays, the concentrations of the active peptide were set to 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively. The detection methods for the inhibition rates of PTP1B, DPP-IV, α-glucosidase, and α-amylase were all as described in Example 1. The detection results are as follows: Figure 7 As shown, FDY, DLGF, DGWR, and ECMFPK can all effectively inhibit the activities of PTP1B, DPP-IV, α-glucosidase, and α-amylase. Moreover, the inhibitory effect on the activities of PTP1B, DPP-IV, α-glucosidase, and α-amylase is enhanced with the increase of active peptide concentration.

[0157] The IC50 values ​​of the PTP1B inhibition rates of four bioactive peptides—FDY, DLGF, DGWR, and ECMFPK—were calculated. 50 The concentrations were 0.8291 mg / mL, 1.247 mg / mL, 0.03766 mg / mL, and 0.3345 mg / mL, respectively. The IC50 values ​​for the DPP-IV inhibition rates of the four active peptides FDY, DLGF, DGWR, and ECMFPK were also presented. 50 The IC50 values ​​were 1.187 mg / mL, 0.8934 mg / mL, 1.429 mg / mL, and 0.5820 mg / mL, respectively. The IC50 values ​​for the α-glucosidase inhibition rates of the four bioactive peptides FDY, DLGF, DGWR, and ECMFPK were also determined. 50 The IC50 values ​​were 1.153 mg / mL, 0.9524 mg / mL, 0.8182 mg / mL, and 1.271 mg / mL, respectively. The IC50 values ​​for the α-amylase inhibition rates of the four bioactive peptides FDY, DLGF, DGWR, and ECMFPK were also presented. 50 The concentrations were 1.363 mg / mL, 0.7495 mg / mL, 1.209 mg / mL, and 1.613 mg / mL, respectively.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a complex of active peptides from Alaska pollock bone, characterized in that, Includes the following steps: The bones of the pollock were initially hydrolyzed using flavor protease.

2. The method for preparing the Alaska pollock bone active peptide complex according to claim 1, characterized in that, Ultrafiltration was performed on the products after enzymatic hydrolysis of flavor proteases, selecting components with a molecular weight <3 kDa.

3. The method for preparing the Alaska pollock bone active peptide complex according to claim 2, characterized in that, For components with a molecular weight <3kDa, targeted enzymatic hydrolysis was performed using trypsin.

4. The method for preparing the Alaska pollock bone active peptide complex according to claim 3, characterized in that, The conditions for targeted trypsin hydrolysis include: pH 8.0, trypsin addition of 4000 U / g, hydrolysis at 37℃ for 2.5 h; and enzyme inactivation after trypsin hydrolysis.

5. The method for preparing the Alaska pollock bone active peptide complex according to any one of claims 1-4, characterized in that, The conditions for flavor protease hydrolysis include: pH 7.5, flavor protease addition of 5000 U / g, hydrolysis at 50℃ for 4 h; and enzyme inactivation after flavor protease hydrolysis.

6. The method for preparing the Alaska pollock bone active peptide complex according to any one of claims 1-4, characterized in that, It also includes a freeze-drying step.

7. Alaska pollock bone active peptides, characterized in that, This includes one or more of FDY, DLGF, DGWR, and ECMFPK.

8. A method for preparing active peptides from Alaska pollock bone, characterized in that, Includes the following steps: Solid-phase synthesis of the pollock bone active peptide of claim 7.

9. The use of the Alaska pollock bone active peptide complex in any one or more of (1) to (6), wherein the Alaska pollock bone active peptide complex is prepared by the preparation method described in any one of claims 1-6. (1) Preparation of hypoglycemic products; (2) To prepare products that improve insulin resistance; (3) Preparation of PTP1B inhibitors; (4) Preparation of DPP-IV inhibitors; (5) Preparation of α-glucosidase inhibitors; (6) Preparation of α-amylase inhibitors.

10. The use of the Alaska pollock bone active peptide according to claim 7 in any one or more of (1) to (6), (1) Preparation of hypoglycemic products; (2) To prepare products that improve insulin resistance; (3) Preparation of PTP1B inhibitors; (4) Preparation of DPP-IV inhibitors; (5) Preparation of α-glucosidase inhibitors; (6) Preparation of α-amylase inhibitors.

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