Pancreatic lipase inhibitory peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioactive peptides, and particularly relates to a pancreatic lipase inhibitory peptide as well as a preparation method and application thereof. The pancreatic lipase inhibitory peptide provided by the invention comprises at least one of oligopeptides of which the amino acid sequences are phenylalanine-glycine-phenylalanine, leucine-leucine-tyrosine, leucine-tyrosine-leucine, phenylalanine-alanine-proline and phenylalanine-tryptophan-glycine. The pancreatic lipase inhibitory peptide can effectively inhibit the activity of pancreatic lipase, can be used for development of common food and products such as health food and drugs related to fat reduction, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and particularly relates to a pancreatic lipase inhibitory peptide, a preparation method thereof and an application thereof. Background Art

[0002] Obesity is a global public health problem, mainly a metabolic disorder caused by unbalanced diet and excessive fat accumulation, especially closely related to elevated triglyceride levels. It is a key cause of various prevalent chronic diseases, including dyslipidemia, diabetes, and cardiovascular diseases, etc.

[0003] Pancreatic lipase (PL) is a key enzyme for lipid hydrolysis, mainly decomposing 50% - 70% of dietary fats present in the small intestine into glycerol and fatty acids, and is an effective target for obesity prevention measures. Therefore, effectively inhibiting the activity of PL to limit the entry of free fatty acids generated during lipid hydrolysis into the bloodstream for fat synthesis is considered a promising method for weight loss and prevention of obesity-related diseases. Currently, drugs commonly used to inhibit the activity of PL, such as orlistat, although proven effective in controlling obesity, also cause various side effects, including diarrhea, flatulence, and fatty stools, and long-term use brings strong discomfort. In addition, the relatively high price of these drugs. Therefore, there is an urgent need to continue to search for new PL inhibitors with higher cost-effectiveness and lower side effects to assist in the treatment of obesity. Summary of the Invention

[0004] Aiming at the above technical problems, the present invention provides a pancreatic lipase inhibitory peptide, a preparation method thereof and an application thereof. The pancreatic lipase inhibitory peptide provided by the present invention has excellent pancreatic lipase inhibitory activity, and the preparation method is relatively simple and controllable, and can be used for the development of ordinary foods, health foods or drugs, and has broad application prospects.

[0005] To achieve the above invention object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a pancreatic lipase inhibitory peptide, which contains at least one of oligopeptides with amino acid sequences of phenylalanine - glycine - phenylalanine (abbreviated as FGF), leucine - leucine - tyrosine (abbreviated as LLY), leucine - tyrosine - leucine (abbreviated as LYL), phenylalanine - alanine - proline (abbreviated as FAP), and phenylalanine - tryptophan - glycine (abbreviated as FWG).

[0006] Experiments have proven that the above-mentioned oligopeptides have excellent inhibitory activity against pancreatic lipase. Therefore, they can effectively limit the free fatty acids generated during lipid hydrolysis from entering the bloodstream for fat synthesis, thereby achieving the purpose of preventing and treating obesity and related metabolic syndromes. Moreover, the above-mentioned oligopeptides are all derived from food proteins. On the one hand, they have higher safety compared to chemical drugs, and the Toxinpred toxicity verification results show that there is no risk of toxicity accumulation. On the other hand, they can still maintain more than 80% of their activity after gastrointestinal digestion, thus ensuring that they can still play a role after digestion.

[0007] In addition, the molecular weights of the above-mentioned oligopeptides FGF, LLY, LYL, FAP, and FWG are 372.44 Da, 410.53 Da, 410.53 Da, 336.41 Da, and 411.47 Da respectively. The relatively small molecular weights endow them with excellent membrane permeability and water solubility, making them more easily formulated into various products. And the tripeptide structure enables them to more easily target and bind to the catalytic triad or substrate-binding domain of pancreatic lipase through hydrogen bonding, hydrophobic, and electrostatic interactions, forming a unique non-competitive reversible inhibition mode, making them unaffected by substrate concentration and still being able to inhibit fat decomposition for a long time in the complex intestinal environment.

[0008] Therefore, the application scenarios of this pancreatic lipase inhibitory peptide can cover four major fields: ordinary foods, health foods, pharmaceutical adjuvants, and cosmetics, providing a solution with both high efficiency and safety for the natural intervention of obesity and related metabolic syndromes, and having significant potential for industrial transformation.

[0009] The second aspect of the present invention provides a preparation method of a pancreatic lipase inhibitory peptide, which specifically includes the following steps: S1. Prepare sea cucumber homogenate by mixing sea cucumber and water at a material-liquid ratio of 1:(18 - 20). First, enzymatically hydrolyze with pepsin, and then enzymatically hydrolyze with subtilisin. After the enzymatic hydrolysis is completed, inactivate the enzyme, and add ethanol to the obtained enzymatic hydrolysate to remove sugars, obtaining a sea cucumber protease hydrolysate; S2. Ultrafilter the sea cucumber protease hydrolysate obtained in S1 using gel chromatography, collect the fraction with a molecular weight less than 1 kDa, and separate and purify the obtained fraction using preparative reverse high-performance liquid chromatography to obtain the pancreatic lipase inhibitory peptide.

[0010] This preparation method combines traditional enzymatic hydrolysis technology and modern separation technology. By sequentially enzymatically hydrolyzing with pepsin and subtilisin, the prepared pancreatic lipase inhibitory peptide contains the above-mentioned oligopeptides FGF, LLY, LYL, FAP, and FWG.

[0011] Compared with the synthesis method, this preparation method provides a safer, more economical, and greener solution for the preparation of pancreatic lipase inhibitory peptides, and has the feasibility of large-scale production.

[0012] Preferably, the sea cucumbers in S1 are selected from dried sea cucumbers or fresh sea cucumbers. After removing the internal organs, surface dirt, and salt, and repeatedly soaking and changing water to reduce the impurity content, they can be used as raw materials.

[0013] Preferably, the material-liquid ratio in S1 is 1:20.

[0014] Preferably, the addition amount of pepsin for enzymatic hydrolysis in S1 is 1500 - 2500 U / g of dried sea cucumbers, the enzymatic hydrolysis pH is 2.4 - 2.6, the enzymatic hydrolysis temperature is 35 - 39 °C, and the enzymatic hydrolysis time is 1 - 2 h. The further preferred enzymatic hydrolysis pH is 2.5, and the enzymatic hydrolysis temperature is 37 °C.

[0015] Preferably, the addition amount of Bacillus subtilis protease for enzymatic hydrolysis in S1 is 4000 - 8000 U / g of dried sea cucumbers, the enzymatic hydrolysis pH is 7.9 - 8.1, the enzymatic hydrolysis temperature is 43 - 47 °C, and the enzymatic hydrolysis time is 3 - 3.5 h. The further preferred enzymatic hydrolysis pH is 8.0, and the enzymatic hydrolysis temperature is 45 °C.

[0016] Preferably, the method for inactivating enzymes in S1 is to inactivate enzymes at 95 - 100 °C for 10 - 12 min. The further preferred method for inactivating enzymes is to inactivate enzymes at 100 °C for 10 min.

[0017] Preferably, the volume concentration of ethanol after adding ethanol in S1 is 75% - 85%. The further preferred volume concentration of ethanol is 80%.

[0018] Preferably, the chromatographic column for gel chromatography in S2 is TK-Col TA-GF30, the detection wavelength of the ultraviolet detector is 220 nm, and the equilibration buffer is ultrapure water.

[0019] More preferably, the injection concentration is 10 mg / mL.

[0020] Preferably, the chromatographic conditions for preparative reversed-phase high-performance liquid chromatography in S2 include: the chromatographic column is AgilentPrep C18, with specifications of 100 Å, 5 µm, 30×100 mm, the detection wavelength is 220 nm, the column temperature is 30 °C, mobile phase A is an aqueous solution of 0.05% - 0.1% trifluoroacetic acid (TFA), mobile phase B is an aqueous acetonitrile solution containing 0.05% - 0.1% trifluoroacetic acid, and the volume percentage concentration of acetonitrile in the aqueous acetonitrile solution is 84% - 85%; the elution gradient, the elution program is 96% - 50%A from 0 - 50 min; 50% - 0%A from 50 - 54 min; 0%A from 54 - 60 min; collect the effluent from 8 - 12 min.

[0021] Further preferably, the mobile phase A is an aqueous solution of 0.1% trifluoroacetic acid, and the mobile phase B is an acetonitrile aqueous solution containing 0.1% trifluoroacetic acid, and the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 84%.

[0022] The third aspect of the present invention provides the use of the above-mentioned pancreatic lipase inhibitory peptide or the pancreatic lipase inhibitory peptide prepared by the above-mentioned preparation method in the preparation of food.

[0023] The fourth aspect of the present invention provides the use of the above-mentioned pancreatic lipase inhibitory peptide or the pancreatic lipase inhibitory peptide prepared by the above-mentioned preparation method in the preparation of health foods that help control body fat.

[0024] The fifth aspect of the present invention provides the use of the above-mentioned pancreatic lipase inhibitory peptide or the pancreatic lipase inhibitory peptide prepared by the above-mentioned preparation method in the preparation of pharmaceutical adjuvants for the treatment and / or prevention of obesity, and the active ingredient includes the above-mentioned pancreatic lipase inhibitory peptide or the pancreatic lipase inhibitory peptide prepared by the above-mentioned preparation method. The pharmaceutical adjuvant can be used alone for the treatment and / or prevention of obesity, or can be used in combination with other drugs for the treatment or prevention of obesity to enhance the curative effect.

[0025] The sixth aspect of the present invention provides the use of the above-mentioned pancreatic lipase inhibitory peptide or the pancreatic lipase inhibitory peptide prepared by the above-mentioned preparation method in the preparation of external products for reducing subcutaneous fat, and the active ingredient includes the above-mentioned pancreatic lipase inhibitory peptide or the pancreatic lipase inhibitory peptide prepared by the above-mentioned preparation method. The external products include slimming creams and the like.

[0026] The beneficial effects of the present invention are as follows: (1) The amino acid sequences in the pancreatic lipase inhibitory peptide provided by the present invention are all derived from food proteins. Through toxicity verification by Toxinpred, there is no risk of toxicity accumulation, and the long-term use safety is significantly better than that of chemically synthesized inhibitors (such as orlistat), and it can effectively avoid side effects such as intestinal discomfort that may be caused by chemically synthesized inhibitors; moreover, since the amino acid sequences of the above-mentioned oligopeptides are derived from food proteins, they can still maintain more than 80% of their activity after gastrointestinal digestion; (2) Experiments have confirmed that the above-mentioned oligopeptides FGF, LLY, LYL, FAP, and FWG have excellent inhibitory activities against pancreatic lipase, and the half-inhibitory concentrations can reach 3.579 mmol / L, 6.308 mmol / L, 8.539 mmol / L, 9.278 mmol / L, and 11.860 mmol / L respectively, and can effectively limit the free fatty acids generated during lipid hydrolysis from entering the bloodstream to synthesize fat; (3)All the oligopeptides in the pancreatic lipase inhibitory peptide provided by the present invention are tripeptides, with a simple structure, which can be directly prepared by artificial synthesis. Moreover, the tripeptide structure enables it to specifically bind to the catalytic triad (Ser152-Asp176-His263) or the substrate-binding domain of pancreatic lipase, forming a non-competitive reversible inhibition. The non-competitive inhibition mechanism makes it unaffected by the substrate concentration and still able to inhibit fat decomposition for a long time in the complex intestinal environment. In addition, compared with other pancreatic lipase inhibitory peptides, the above-mentioned oligopeptides have a smaller molecular weight, so they can be directly absorbed by the intestine and act in the body, with higher utilization rate; (4)The above-mentioned oligopeptides have good water solubility and can be directly added to solid beverages (such as meal replacement powders), dairy products or nutrition bars without affecting the taste of the food; they can also be used in combination with existing anti-obesity drugs to reduce the burden on the liver and kidneys by reducing the drug dosage; they can also support the development of cosmetics such as slimming creams to inhibit local fat accumulation; (5)The preparation method of the pancreatic lipase inhibitory peptide provided by the present invention extracts the above-mentioned oligopeptides from sea cucumbers through enzymatic hydrolysis technology. The concept of green bio-manufacturing is implemented throughout the process from raw material screening to final product preparation, which is safer and the cost is lower than that of the chemical synthesis route, and has the economic feasibility for large-scale production; (6)The present invention broadens the commercial application dimension of sea cucumber polypeptides and is of great significance for the development of safer health foods that help control body fat. Description of the Drawings

[0027] Figure 1 It is the high performance liquid chromatography (HPLC) diagram of the oligopeptide FGF synthesized by the solid-phase method in Example 4 of the present invention; Figure 2 It is the mass spectrometry diagram of the oligopeptide FGF synthesized by the solid-phase method in Example 4 of the present invention; Figure 3 It is the high performance liquid chromatography (HPLC) diagram of the oligopeptide LLY synthesized by the solid-phase method in Example 5 of the present invention; Figure 4 It is the mass spectrometry diagram of the oligopeptide LLY synthesized by the solid-phase method in Example 5 of the present invention; Figure 5 It is the high performance liquid chromatography (HPLC) diagram of the oligopeptide LYL synthesized by the solid-phase method in Example 6 of the present invention; Figure 6 It is the mass spectrometry diagram of the oligopeptide LYL synthesized by the solid-phase method in Example 6 of the present invention; Figure 7 It is the high performance liquid chromatography (HPLC) diagram of the oligopeptide FAP synthesized by the solid-phase method in Example 7 of the present invention; Figure 8 It is the mass spectrometry diagram of the oligopeptide FAP synthesized by the solid-phase method in Example 7 of the present invention; Figure 9It is the high-performance liquid chromatography (HPLC) chromatogram of the oligopeptide FWG synthesized by the solid-phase method in Example 8 of the present invention; Figure 10 It is the mass spectrometry (MS) spectrum of the oligopeptide FWG synthesized by the solid-phase method in Example 8 of the present invention; Figure 11 It is the global molecular docking image and molecular interaction diagram of the oligopeptide FGF and pancreatic lipase in Test Example 1 of the present invention; Figure 12 It is the global molecular docking image and molecular interaction diagram of the oligopeptide LLY and pancreatic lipase in Test Example 1 of the present invention; Figure 13 It is the global molecular docking image and molecular interaction diagram of the oligopeptide LYL and pancreatic lipase in Test Example 1 of the present invention; Figure 14 It is the global molecular docking image and molecular interaction diagram of the oligopeptide FAP and pancreatic lipase in Test Example 1 of the present invention; Figure 15 It is the global molecular docking image and molecular interaction diagram of the oligopeptide FWG and pancreatic lipase in Test Example 1 of the present invention; Figure 16 It is the determination result of the IC 50 value of pancreatic lipase inhibitory activity in Test Example 2 of the present invention. Detailed implementation manners

[0028] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0029] PL is a key enzyme for lipid hydrolysis and an effective target for obesity prevention measures. Effectively inhibiting the activity of PL is expected to be a method for reducing body weight and preventing obesity-related diseases. Although the drugs commonly used to inhibit the activity of PL can effectively control obesity, they also cause various side effects. Bioactive peptides derived from food usually have the characteristics of being natural, safe and easy to obtain, and are often used in the development of ordinary foods and health foods. Sea cucumbers are rich in protein and have a complete variety of amino acids, making them a good source of marine bioactive peptides. Existing studies have shown that sea cucumber peptides have biological activities such as anti-fatigue and enhancing physical fitness, cardiovascular protection, antioxidant and anti-inflammatory effects, immune regulation and anti-tumor adjuvant effects, and promoting wound healing, but it has not been found that sea cucumber peptides can be used to inhibit the activity of pancreatic lipase.

[0030] The present invention has obtained a pancreatic lipase inhibitory peptide from sea cucumber polypeptides through experimental research. The pancreatic lipase inhibitory peptide contains at least one of the oligopeptides with amino acid sequences of phenylalanine-glycine-phenylalanine, leucine-leucine-tyrosine, leucine-tyrosine-leucine, phenylalanine-alanine-proline and phenylalanine-tryptophan-glycine. The above oligopeptides have excellent inhibitory activity against pancreatic lipase, and are all derived from food proteins with high safety; they have small molecular weight, are easier to absorb, and can target and bind to the pancreatic lipase catalytic triad or substrate binding domain to form a non-competitive reversible inhibition mode, thereby having a long-term fat control advantage.

[0031] The present invention also provides a method for preparing a pancreatic lipase inhibitory peptide, which specifically comprises the following steps: S1. Prepare sea cucumber homogenate by mixing sea cucumber and water at a material-liquid ratio of 1:(18-20), first enzymolyze with pepsin, and then enzymolyze with Bacillus subtilis protease; after the enzymolysis is completed, inactivate the enzyme, add ethanol to the obtained enzymolyzate to remove sugar, and obtain sea cucumber protease hydrolyzate; S2. Ultrafiltering the sea cucumber protein hydrolysate obtained in S1 using gel chromatography to collect components with a molecular weight less than 1 kDa, and separating and purifying the obtained components using preparative reversed-phase high performance liquid chromatography to obtain the pancreatic lipase inhibitory peptide.

[0032] The scheme of the present invention is described below through specific embodiments.

[0033] The porcine pancreatic lipase (type II) used in the following examples was purchased from Sigma-Aldrich (Cat. No. L3126-25G), p-nitrophenylbutyrate (p-NPB) was purchased from MacLean Chemical Reagent Co., Ltd., 1M Tris-HCl solution was purchased from Beijing Lanjieke Technology Co., Ltd., and dimethyl sulfoxide (DMSO) was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd. Oligopeptides were synthesized at Hefei Sener Biotechnology Co., Ltd.

[0034] Unless otherwise specified in the following examples, all solutions mentioned use water as the solvent.

[0035] The preparation method of the solution used in the following examples is: 50 mM Tris-HCl solution: Take 1 mL of 1 M Tris-HCl solution and add 19 mL of distilled water; 0.25 mg / mL pancreatic lipase solution: Weigh 100 mg of porcine pancreatic lipase, add 10 mL of 50 mM Tris-HCl (pH 8.0) solution, and shake until uniform to prepare a 10 mg / mL pancreatic lipase solution; take 1 mL from it and add 9 mL of 50 mM Tris-HCl (pH 8.0) solution to prepare a 1 mg / mL pancreatic lipase solution; repeat the above dilution until the concentration is 0.25 mg / mL; p-Nitrophenyl butyrate (p-NPB) solution: Weigh 0.0523 g of p-nitrophenyl butyrate and make up to 25 mL in a brown volumetric flask with absolute ethanol; Simulated gastric fluid (SGF): 34 mM NaCl, 0.15% (w / v) pepsin, adjusted to pH 1.5 - 2.0 with 1 mol / L hydrochloric acid; Simulated intestinal fluid (SIF): 50 mM KH2PO4 buffer, 10 mM sodium taurocholate (bile salt), 1.0 mg / mL pancreatin, adjusted to pH 7.0 ± 0.2 with 1 mol / L NaOH; Termination solution: 0.5 mM Pepstatin A (pepsin inhibitor), 1 mM PMSF (serine protease inhibitor).

[0036] Unless otherwise specified, the raw materials, reagents, drugs or instruments used in the following examples are all conventional commercially available products obtained through commercial channels. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art.

[0037] Example 1 This example provides a pancreatic lipase inhibitory peptide and its preparation method.

[0038] (1) Raw material pretreatment: Select soaked dried sea cucumbers as raw materials, remove the internal organs, surface dirt and salt, and soak and change water repeatedly to reduce the impurity content.

[0039] (2) Enzymatic hydrolysis: Make a sea cucumber homogenate with sea cucumbers and water at a material-liquid ratio of 1:20, select pepsin for preliminary hydrolysis at 2000 U / g under acidic conditions, the enzymatic hydrolysis parameters are pH 2.5, enzymatic hydrolysis temperature 37 °C, enzymatic hydrolysis time 2 h, then adjust the pH to 8.0 with 50 mM Tris-HCl solution, add subtilisin protease at 6000 U / g and enzymatically hydrolyze at 45 °C for 3 h, and terminate the reaction in a boiling water bath for 10 min.

[0040] (3) Debriding: Centrifuge the enzymatic hydrolysate obtained in step (1) at a rotational speed of 8000 r / min for 15 min. Then, take the supernatant and perform rotary evaporation. After concentrating it to 1 / 5 of the original volume, slowly add four times the volume of absolute ethanol. Mix well and let it stand at 4°C for 72 h. After centrifuging again, rotary evaporate and concentrate the supernatant, and then freeze-dry to obtain the sea cucumber protease hydrolysate.

[0041] (4) Gel chromatography separation: Dissolve the sea cucumber protease hydrolysate obtained in step (3) in ultrapure water, centrifuge (10000×g, 20 min) to remove insoluble particles, take the supernatant and use a 3 kDa ultrafiltration tube to separate small peptides, and then use gel chromatography for separation and purification. Collect the effluent with a molecular weight less than 1 kDa. Among them, the chromatographic column for gel chromatography is TK-Col TA-GF30 (1.6 cm×60 cm), the detection wavelength of the ultraviolet detector is 220 nm, the equilibrium buffer is ultrapure water, the injection concentration is 10 mg / mL, and the injection volume is 1 mL.

[0042] (5) Preparative reverse-phase high-performance liquid chromatography separation (RP-HPLC): Separate and purify the effluent obtained in step (4) using preparative reverse-phase high-performance liquid chromatography to obtain high-purity sea cucumber oligopeptides. Among them, the chromatographic column for preparative reverse-phase high-performance liquid chromatography is Agilent Prep C18 (100Å, 5 µm, 30×100 mm), the detection wavelength is 220 nm, the column oven temperature is 30°C, mobile phase A is 0.1% TFA aqueous solution, and mobile phase B is 0.1% TFA acetonitrile aqueous solution (acetonitrile concentration is 84%); elution gradient, the elution program is 96%-50%A for 0 - 50 min; 50% - 0%A for 50 - 54 min; 0%A for 54 - 60 min; collect the effluent from 8 to 12 min.

[0043] (6) Drying: Concentrate the effluent obtained in step (5) to an appropriate amount and then freeze-dry to obtain sea cucumber oligopeptide powder with good solubility and good pancreatic lipase inhibitory activity.

[0044] (7) Inhibition rate determination: Prepare a polypeptide solution with the sea cucumber oligopeptide powder obtained in step (6) using a DMSO (<1%) solution for the determination of the inhibitory rate of pancreatic lipase activity (the method is the same as in Test Example 2). The solution concentration is 15 mg / mL, and the measured inhibition rate is 40.12%.

[0045] (8)Isolation and identification of bioactive peptide segments: The sea cucumber oligopeptide powder obtained in step (6) was subjected to chromatographic separation. The solution A used in liquid chromatography was 0.1% formic acid aqueous solution, and the solution B was 0.1% formic acid acetonitrile aqueous solution (acetonitrile was 84%). The liquid chromatography column (0.15 mm × 150 mm, RP-C18, Column Technology Inc.) was equilibrated with 95% of solution A. The sample was loaded onto Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) by an autosampler, and then separated by the liquid chromatography column. The relevant liquid phase gradient settings were as follows: 0 min → 50 min, the linear gradient of solution B was from 4% to 50%; 50 min → 54 min, the linear gradient of solution B was from 50% to 100%; 54 min → 60 min, solution B was maintained at 100%.

[0046] After the enzymatic hydrolysate was separated by capillary high performance liquid chromatography, it was subjected to mass spectrometry analysis using a Q Exactive HF-X mass spectrometer (ThermoFisher). Analysis duration: 60 min. Detection mode: positive ion. The mass-to-charge ratios of polypeptides and polypeptide fragments were collected according to the following method: 10 fragment spectra (MS2 scan) were collected after each full scan, and finally 1403 polypeptide fragments were obtained, including oligopeptides FGF, LLY, LYL, FAP, and FWG.

[0047] Example 2 This example provides a pancreatic lipase inhibitory peptide and its preparation method.

[0048] (1) Raw material pretreatment: Fresh sea cucumbers were selected as raw materials, and the internal organs, surface dirt, and salts were removed. They were repeatedly soaked and changed water to reduce the impurity content.

[0049] (2) Enzymatic hydrolysis: The sea cucumbers and water were made into a sea cucumber homogenate according to a material-liquid ratio of 1:20. Pepsin was selected for preliminary hydrolysis under acidic conditions at 1500 U / g. The enzymatic hydrolysis parameters were pH 2.4, enzymatic hydrolysis temperature 35 °C, and enzymatic hydrolysis time 2 h. Then the pH was adjusted to 7.9 with 50 mM Tris-HCl solution, and subtilisin protease was added at 4000 U / g for enzymatic hydrolysis at 43 °C for 3.5 h. The reaction was terminated by boiling water bath for 10 min.

[0050] (3) Debloating: The same as Example 1.

[0051] (4) Gel chromatography separation: Dissolve the sea cucumber protease hydrolysate obtained in step (3) in ultrapure water, centrifuge (8000×g, 20 min) to remove insoluble particles, take the supernatant and use a 3 kDa ultrafiltration tube to separate small peptides, and then use gel chromatography for separation and purification, and collect the effluent with a molecular weight less than 1 kDa. Among them, the chromatographic column for gel chromatography is TK-Col TA-GF30 (1.6 cm×60 cm), the detection wavelength of the ultraviolet detector is 220 nm, the equilibrium buffer is ultrapure water, the injection concentration is 10 mg / mL, and the injection volume is 1 mL.

[0052] (5) Preparative reverse-phase high performance liquid chromatography separation: Use preparative reverse-phase high performance liquid chromatography to separate and purify the effluent obtained in step (4) to obtain high-purity sea cucumber oligopeptides. Among them, the chromatographic column for preparative reverse-phase high performance liquid chromatography is Agilent Prep C18 (100Å, 5 µm, 30×100 mm), the detection wavelength is 220 nm, the column oven temperature is 30 °C, mobile phase A is 0.1% TFA aqueous solution, and mobile phase B is 0.05% TFA acetonitrile aqueous solution (acetonitrile concentration is 85%); elution gradient, the elution program is 96%-50% A for 0 - 50 min; 50% - 0% A for 50 - 54 min; 0% A for 54 - 60 min; collect the effluent from 8 - 12 min.

[0053] (6) Drying: The same as in Example 1. The obtained sea cucumber oligopeptide powder contains FGF, LLY, LYL, FAP, and FWG.

[0054] (7) Separation of bioactive peptide segments: Perform chromatographic separation on the sea cucumber oligopeptide powder obtained in step (6), and the chromatographic conditions are the same as in Example 1. Collect the effluent according to the elution times of FGF, LLY, LYL, FAP, and FWG to obtain high-purity FGF, LLY, LYL, FAP, and FWG.

[0055] Example 3 This example provides a pancreatic lipase inhibitory peptide and its preparation method.

[0056] (1) Raw material pretreatment: Select the soaked dried sea cucumbers as raw materials, remove the internal organs, surface dirt, and salt, and soak and change water repeatedly to reduce the impurity content.

[0057] (2) Enzymatic hydrolysis: Sea cucumbers and water were made into sea cucumber homogenate according to a material-liquid ratio of 1:20. Pepsin was selected for preliminary hydrolysis under acidic conditions at 2500 U / g. The enzymatic hydrolysis parameters were pH 2.6, enzymatic hydrolysis temperature 39 °C, and enzymatic hydrolysis time 1 h. Then, the pH was adjusted to 8.1 with 50 mM Tris-HCl solution, and subtilisin was added at 8000 U / g for enzymatic hydrolysis at 47 °C for 3 h. The reaction was terminated by maintaining it in a 95 °C water bath for 12 min.

[0058] (3) Debloating: The same as Example 1.

[0059] (4) Gel chromatography separation: The same as Example 1.

[0060] (5) Preparative reversed-phase high-performance liquid chromatography separation (RP-HPLC): The effluent obtained in step (4) was separated and purified by preparative reversed-phase high-performance liquid chromatography to obtain high-purity sea cucumber oligopeptides. Among them, the chromatographic column of the preparative reversed-phase high-performance liquid chromatography was Agilent Prep C18 (100 Å, 5 µm, 30×100 mm), the detection wavelength was 220 nm, the column oven temperature was 30 °C, mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.05% TFA acetonitrile aqueous solution (acetonitrile concentration was 84%); the elution gradient and elution program were 96%-50% A for 0 - 50 min; 50% - 0% A for 50 - 54 min; 0% A for 54 - 60 min; the effluent from 8 - 12 min was collected.

[0061] (6) Drying: The same as Example 1. The obtained sea cucumber oligopeptide powder contains FGF, LLY, LYL, FAP, and FWG.

[0062] (7) Separation of active peptide segments: The sea cucumber oligopeptide powder obtained in step (6) was chromatographically separated. The chromatographic conditions were the same as in Example 1. The effluent was collected according to the elution times of FGF, LLY, LYL, FAP, and FWG, and high-purity FGF, LLY, LYL, FAP, and FWG were obtained.

[0063] Example 4 This example provides an oligopeptide FGF with pancreatic lipase inhibitory activity.

[0064] Using the conventional solid-phase synthesis method, a polypeptide was synthesized in the amino acid sequence of phenylalanine-glycine-phenylalanine, and the oligopeptide FGF was obtained. It was detected by LC-MS / MS. The chromatographic and mass spectrometric conditions were the same as in Test Example 1. The obtained HPLC chromatogram is as Figure 1 shown, and the mass spectrum is as Figure 2 shown.

[0065] Example 5 This example provides an oligopeptide LLY with pancreatic lipase inhibitory activity.

[0066] Using the conventional solid-phase synthesis method, a polypeptide was synthesized in the amino acid sequence of leucine-leucine-tyrosine, and the oligopeptide LLY was obtained. It was detected by LC-MS / MS, and the chromatographic and mass spectrometric conditions were the same as those in Test Example 1. The obtained HPLC chromatogram is as Figure 3 shown, and the mass spectrum is as Figure 4 shown.

[0067] Example 6 This example provides an oligopeptide LYL with pancreatic lipase inhibitory activity.

[0068] Using the conventional solid-phase synthesis method, a polypeptide was synthesized in the amino acid sequence of leucine-tyrosine-leucine, and the oligopeptide LYL was obtained. It was detected by LC-MS / MS, and the chromatographic and mass spectrometric conditions were the same as those in Test Example 1. The obtained HPLC chromatogram is as Figure 5 shown, and the mass spectrum is as Figure 6 shown.

[0069] Example 7 This example provides an oligopeptide FAP with pancreatic lipase inhibitory activity.

[0070] Using the conventional solid-phase synthesis method, a polypeptide was synthesized in the amino acid sequence of phenylalanine-alanine-proline, and the oligopeptide FAP was obtained. It was detected by LC-MS / MS, and the chromatographic and mass spectrometric conditions were the same as those in Test Example 1. The obtained HPLC chromatogram is as Figure 7 shown, and the mass spectrum is as Figure 8 shown.

[0071] Example 8 This example provides an oligopeptide FWG with pancreatic lipase inhibitory activity.

[0072] Using the conventional solid-phase synthesis method, a polypeptide was synthesized in the amino acid sequence of phenylalanine-tryptophan-glycine, and the oligopeptide FWG was obtained. It was detected by LC-MS / MS, and the chromatographic and mass spectrometric conditions were the same as those in Test Example 1. The obtained HPLC chromatogram is as Figure 9 shown, and the mass spectrum is as Figure 10 shown.

[0073] Example 9 This example provides a pancreatic lipase inhibitory peptide, which is compounded from the oligopeptides in Examples 4 to 8 in a mass ratio of 1:1:1:1:1.

[0074] Test Example 1 This test example predicted the safety and activity of the oligopeptides FGF, LLY, LYL, FAP, and FWG obtained in Example 1.

[0075] Predicted by ToxinPred, the oligopeptides FGF, LLY, LYL, FAP, and FWG are non-toxic and non-carcinogenic.

[0076] Predicted by gastrointestinal absorption ability and Lipinski's five rules, the oligopeptides FGF, LLY, LYL, FAP, and FWG have good gastrointestinal absorption ability and high-potential biological activity.

[0077] Download the 3D structure of pancreatic lipase (PDB ID: 1LPB) from the RCSB pdb database and perform structure preprocessing through Pymol, including adding hydrogen atoms, atom dehybridization, deleting water molecules, and removing the original ligands on the protein molecule. Construct 3D models of the oligopeptides FGF, LLY, LYL, FAP, and FWG using the Pymol software. Use the AutoDock Vina 1.1.2.0 (the Scripps Research Institute, La Jolla, CA, USA) software for molecular docking operations and perform visualization processing using the Pymol software. The docking engine is selected as AutoDock Vina, and the docking box size is set to 18.0 × 19.3 × 16.6 Å3, with the center point coordinates (x, y, z) = (4.1, 27.7, 48.7).

[0078] The docking results are as Figures 11 to 15 shown. Phenylalanine-glycine-phenylalanine (FGF) forms 6 hydrogen bonds with SER152, HIS151, HIS263, GIY76, PHE77, and ASP79A of pancreatic lipase, and there are 4 types of interactions; leucine-leucine-tyrosine (LLY) forms 3 hydrogen bonds with HIS151, HIS263, and GLY76 of pancreatic lipase, forms 1 carbon-hydrogen bond, and there are 4 types of interactions; leucine-tyrosine-leucine (LYL) forms 4 hydrogen bonds with PHE77, ASP79, HIS151, and GLY76 of pancreatic lipase, and there are 4 types of interactions; phenylalanine-alanine-proline (FAP) forms 1 hydrogen bond with SER152 of pancreatic lipase, and there are 4 types of interactions; phenylalanine-tryptophan-glycine (FWG) forms 5 hydrogen bonds with PHE77, PHE215, ARG256, and ASP79 of pancreatic lipase, forms 1 carbon-hydrogen bond, and there are 4 types of interactions.

[0079] The binding energies of orlistat and the above oligopeptides to pancreatic lipase inhibition are shown in Table 1.

[0080] Table 1 Binding Energies of Active Polypeptides to Pancreatic Lipase Inhibition

[0081] Test Example 2 This test example examined the half-inhibitory concentration of the oligopeptides FGF, LLY, LYL, FAP, and FWG in Examples 4 to 8 on pancreatic lipase.

[0082] Using p-nitrophenyl butyrate (p-NPB) as the substrate, the in vitro inhibitory activity of porcine pancreatic lipase (type II) was measured.

[0083] Sample experimental group: Mix 50 µL of the oligopeptide solution (concentration 100 µM to 10 mM, solvent 0.1% DMSO) with 50 µL of 0.25 mg / mL pancreatic lipase solution in a 96-well microplate, incubate at 25 °C for 15 min, then add 50 µL of p-NPB solution and react in the dark for 10 min, and then measure with a microplate reader.

[0084] Control experimental group: Place 50 µL of 0.25 mg / mL pancreatic lipase solution in a 96-well microplate, incubate at 25 °C for 15 min, then add 50 µL of p-NPB solution and react in the dark for 10 min, and then measure with a microplate reader.

[0085] Sample blank group: Place 50 µL of the oligopeptide solution (concentration 100 µM to 10 mM, solvent 0.1% DMSO) in a 96-well microplate, incubate at 25 °C for 15 min, then add 50 µL of p-NPB solution and react in the dark for 10 min, and then measure with a microplate reader.

[0086] Control blank group: Add 50 µL of p-NPB solution to a 96-well microplate, place in the dark for 10 min, and then measure with a microplate reader.

[0087] Calculate the inhibition rate of pancreatic lipase activity according to the following formula.

[0088]

[0089] In the formula: A1 is the absorbance of the control experimental group; A2 is the absorbance of the sample experimental group; A3 is the absorbance of the control blank group; A4 is the absorbance of the sample blank group.

[0090] Measure the inhibition rate of pancreatic lipase activity of the positive control orlistat by the same method.

[0091] Use Graphpad Prism 9 for fitting analysis and SPSS software for significance analysis to obtain the results as shown in Table 2 and Figure 16 as shown, the half-inhibitory concentration (IC 50) was 0.976 mmol / L, and the IC 50 values of oligopeptide FGF, oligopeptide LLY, oligopeptide LYL, oligopeptide FAP, and oligopeptide FWG were 3.579 mmol / L, 6.308 mmol / L, 8.539 mmol / L, 9.278 mmol / L, and 11.860 mmol / L, respectively.

[0092] Table 2 IC 50 values

[0093] Note: a, b, c, and d represent significant differences between different samples.

[0094] Test Example 3 This test example investigated the biological activities of oligopeptides FGF, LLY, LYL, FAP, and FWG in Examples 4 to 8 and the positive control orlistat in the gastrointestinal tract.

[0095] 1. Simulating gastrointestinal digestion Take 1 mL of the oligopeptide sample (dissolved in SGF buffer) and add it to a 1.5 mL EP tube.

[0096] Add 1 mL of simulated gastric juice (SGF), mix well, and the final volume is 2 mL. Incubate with shaking at 37°C for 2 h. Add 2 μL of 0.5 mM Pepstatin A and adjust the pH to 7.0.

[0097] 2. Intestinal digestion stage Add 2.2 mL of simulated intestinal fluid (SIF) to the gastric digestion product and mix well. Incubate with shaking at 37°C for 2 h, add 10 μL of 1 mM PMSF, mix well, and obtain the digestion termination solution.

[0098] 3. Undigested group: The oligopeptide sample is not treated with SGF / SIF and directly undergoes subsequent activity determination. 4. Treatment of digested samples Transfer the digestion termination solution to an ultrafiltration centrifugal tube (3 kDa MWCO). Centrifuge at 12,000 × g at 4°C for 30 min and collect the filtrate. Dilute the filtrate to an appropriate concentration with 50 mM Tris-HCl solution for activity detection.

[0099] 5. Calculation of inhibition rate Use the measurement method of Test Example 2 to measure the inhibition rate of pancreatic lipase activity, calculate the IC 50 before and after digestion, and compare the activity retention rate.

[0100] Activity retention rate (%) = (IC of undigested group50 / IC after digestion 50 ) × 100%.

[0101] Determine the IC of positive control orlistat before and after digestion by the same method 50 and activity retention rate.

[0102] 6. Results As shown in Table 3, oligopeptides FGF, LLY, LYL, FAP, and FWG can still maintain more than 80% activity after gastrointestinal digestion.

[0103] Table 3 IC before and after digestion 50 and activity retention rate

[0104] Note: a, b, c, d, e represent significant differences between different samples.

[0105] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A pancreatic lipase inhibitory peptide, characterized in that, The pancreatic lipase inhibitory peptide contains at least one of oligopeptides with amino acid sequences of phenylalanine-glycine-phenylalanine, leucine-leucine-tyrosine, leucine-tyrosine-leucine, phenylalanine-alanine-proline, and phenylalanine-tryptophan-glycine.

2. A method for preparing a pancreatic lipase inhibitory peptide, characterized in that, Specifically, it includes the following steps: S1. Prepare a sea cucumber homogenate by mixing sea cucumber and water at a material-liquid ratio of 1:(18 - 20). First, enzymatically hydrolyze it with pepsin, and then with subtilisin. After the enzymatic hydrolysis is completed, inactivate the enzyme, and add ethanol to the obtained enzymatic hydrolysate to remove sugars, obtaining a sea cucumber protease hydrolysate. S2. Ultrafilter the sea cucumber protease hydrolysate obtained in S1 using gel chromatography, collect the fraction with a molecular weight less than 1 kDa, and separate and purify the obtained fraction using preparative reverse high-performance liquid chromatography to obtain the pancreatic lipase inhibitory peptide.

3. The preparation method according to claim 2, characterized in that, In S1, the material-liquid ratio is 1:20; and / or In S1, the addition amount of pepsin for enzymatic hydrolysis with pepsin is 1500 - 2500 U / g of dry sea cucumber, the enzymatic hydrolysis pH is 2.4 - 2.6, the enzymatic hydrolysis temperature is 35 - 39 °C, and the enzymatic hydrolysis time is 1 - 2 h; and / or In S1, the addition amount of subtilisin for enzymatic hydrolysis with subtilisin is 4000 - 8000 U / g of dry sea cucumber, the enzymatic hydrolysis pH is 7.9 - 8.1, the enzymatic hydrolysis temperature is 43 - 47 °C, and the enzymatic hydrolysis time is 3 - 3.5 h; and / or In S1, the method for inactivating the enzyme is to inactivate the enzyme at 95 - 100 °C for 10 - 12 min; and / or In S1, the volume concentration of ethanol after adding ethanol is 75% - 85%.

4. The preparation method according to claim 3, characterized in that, In S1, the pH for enzymatic hydrolysis with pepsin is 2.5, and the enzymatic hydrolysis temperature is 37 °C; and / or In S1, the enzymatic hydrolysis pH for enzymatic hydrolysis with subtilisin is 8.0, and the enzymatic hydrolysis temperature is 45 °C; and / or In S1, the volume concentration of ethanol after adding ethanol is 80%.

5. The preparation method according to any one of claims 2 to 4, characterized in that, In S2, the chromatographic column for gel chromatography is TK-Col TA-GF30, the detection wavelength of the ultraviolet detector is 220 nm, and the equilibration buffer is ultrapure water; and / or The chromatographic conditions for the preparative reverse-phase high-performance liquid chromatography in S2 include: the chromatographic column is Agilent Prep C18, with specifications of 100 Å, 5 µm, 30×100 mm, the detection wavelength is 220 nm, the column temperature is 30 °C, mobile phase A is an aqueous solution of 0.05% - 0.1% trifluoroacetic acid, mobile phase B is an aqueous acetonitrile solution containing 0.05% - 0.1% trifluoroacetic acid, and the volume percentage concentration of acetonitrile in the aqueous acetonitrile solution is 84% - 85%; elution gradient, the elution program is 96% - 50% A for 0 - 50 min; 50% - 0% A for 50 - 54 min; 0% A for 54 - 60 min; collect the effluent from 8 - 12 min.

6. The preparation method according to claim 5, wherein, Mobile phase A is an aqueous solution of 0.1% trifluoroacetic acid, mobile phase B is an aqueous acetonitrile solution containing 0.1% trifluoroacetic acid, and the volume percentage concentration of acetonitrile in the aqueous acetonitrile solution is 84%.

7. Use of the pancreatic lipase inhibitory peptide according to claim 1 or the pancreatic lipase inhibitory peptide prepared by the preparation method according to any one of claims 2 to 6 in the preparation of food.

8. Use of the pancreatic lipase inhibitory peptide according to claim 1 or the pancreatic lipase inhibitory peptide prepared by the preparation method according to any one of claims 2 to 6 in the preparation of health food helpful for controlling body fat.

9. Use of the pancreatic lipase inhibitory peptide according to claim 1 or the pancreatic lipase inhibitory peptide prepared by the preparation method according to any one of claims 2 to 6 in the preparation of a pharmaceutical adjuvant for treating and / or preventing obesity, characterized in that, The active ingredient of the pharmaceutical adjuvant includes the pancreatic lipase inhibitory peptide according to claim 1 or the pancreatic lipase inhibitory peptide prepared by the preparation method according to any one of claims 2 to 6.

10. Use of the pancreatic lipase inhibitory peptide according to claim 1 or the pancreatic lipase inhibitory peptide prepared by the preparation method according to any one of claims 2 to 6 in the preparation of an external product for reducing subcutaneous fat, characterized in that, The active ingredient of the external product includes the pancreatic lipase inhibitory peptide according to claim 1 or the pancreatic lipase inhibitory peptide prepared by the preparation method according to any one of claims 2 to 6.

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