Pancreatic lipase inhibitory peptide, and preparation method and application thereof

Abstract

The present application belongs to the technical field of bioactive peptides, and particularly relates to a pancreatic lipase inhibiting peptide as well as a preparation method and application thereof. The pancreatic lipase inhibiting peptide provided by the present application comprises at least one of the following oligopeptides: phenylalanine-glycine-phenylalanine, leucine-leucine-tyrosine, leucine-tyrosine-leucine, phenylalanine-alanine-proline and phenylalanine-tryptophan-glycine. The pancreatic lipase inhibiting peptide can effectively inhibit the activity of pancreatic lipase, and can be used for the development of common food, health food related to fat reduction, medicine and other products, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, and particularly relates to a pancreatic lipase inhibitory peptide, its preparation method, and its application. Background Technology

[0002] Obesity is a global public health problem, primarily a metabolic disorder caused by an 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 disease.

[0003] Pancreatic lipase (PL) is a key enzyme in lipid hydrolysis, primarily breaking down 50%–70% of dietary fat in the small intestine into glycerol and fatty acids, making it an effective target for obesity prevention. Therefore, effectively inhibiting PL activity to limit the entry of free fatty acids generated during lipid hydrolysis into the bloodstream for liposynthesis is considered a promising approach for weight loss and prevention of obesity-related diseases. Currently used drugs for inhibiting PL activity, such as orlistat, while proven effective in controlling obesity, also cause various side effects, including diarrhea, flatulence, and fatty stools, and long-term use can lead to significant discomfort. Furthermore, these drugs are relatively expensive. Therefore, there is an urgent need to continue searching for novel PL inhibitors with higher cost-effectiveness and fewer side effects to assist in the treatment of obesity. Summary of the Invention

[0004] To address the above-mentioned technical problems, this invention provides a pancreatic lipase inhibitory peptide, its preparation method, and its applications. The pancreatic lipase inhibitory peptide provided by this invention exhibits excellent pancreatic lipase inhibitory activity, and its preparation method is relatively simple and controllable. It can be used in the development of ordinary foods, health foods, or pharmaceuticals, and has broad application prospects.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a pancreatic lipase inhibitory peptide, wherein the pancreatic lipase inhibitory peptide contains at least one of the following oligopeptides with the amino acid sequence 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 demonstrated that the aforementioned oligopeptides exhibit excellent inhibitory activity against pancreatic lipase, thus effectively limiting the entry of free fatty acids generated during lipid hydrolysis into the bloodstream for fat synthesis. This achieves the goal of preventing and treating obesity and related metabolic syndromes. Furthermore, these oligopeptides are all derived from food proteins, which, on the one hand, offer higher safety compared to chemical drugs (Toxinpred toxicity testing results showed no risk of toxicity accumulation); on the other hand, they retain over 80% of their activity after gastrointestinal digestion, ensuring their continued efficacy after digestion.

[0007] In addition, the molecular weights of the aforementioned 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. Their relatively small molecular weight gives them excellent permeabilization and water solubility, making them easier to formulate into various products. Furthermore, their tripeptide structure makes it easier for them to target and bind to the pancreatic lipase catalytic triplet or substrate binding domain through hydrogen bonding, hydrophobicity, and electrostatic interactions, forming a unique non-competitive reversible inhibition mode. This allows them to remain unaffected by substrate concentration and effectively inhibit lipolysis in the complex intestinal environment.

[0008] Therefore, the application scenarios of this pancreatic lipase inhibitory peptide can cover fields such as pharmaceutical excipients and cosmetics, providing a highly efficient and safe solution for natural intervention in obesity and related metabolic syndromes, and has significant potential for industrialization.

[0009] A second aspect of this invention provides a method for preparing pancreatic lipase inhibitory peptides, specifically comprising the following steps: S1. Prepare sea cucumber homogenate by mixing sea cucumber and water at a ratio of 1:(18~20). First, hydrolyze the homogenate with pepsin, then hydrolyze it with Bacillus subtilis protease. After hydrolysis, inactivate the enzymes and add ethanol to the hydrolysate to remove sugar, thus obtaining sea cucumber protease hydrolysate. S2. The sea cucumber protein hydrolysate obtained in S1 is subjected to ultrafiltration using gel chromatography to collect components with a molecular weight less than 1 kDa. The obtained components are then separated and purified using preparative reverse high performance liquid chromatography to obtain the pancreatic lipase inhibitory peptide.

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

[0011] Compared to synthetic methods, this preparation method provides a safer, more economical, and greener solution for the preparation of pancreatic lipase inhibitory peptides, and it is feasible for large-scale production.

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

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

[0014] Preferably, in S1, the amount of pepsin added for hydrolysis is 1500-2500 U / g of dried sea cucumber, the hydrolysis pH is 2.4-2.6, the hydrolysis temperature is 35-39℃, and the hydrolysis time is 1-2 h. More preferably, the hydrolysis pH is 2.5 and the hydrolysis temperature is 37℃.

[0015] Preferably, in S1, the amount of Bacillus subtilis protease added for enzymatic hydrolysis is 4000~8000 U / g dried sea cucumber, the hydrolysis pH is 7.9~8.1, the hydrolysis temperature is 43~47℃, and the hydrolysis time is 3~3.5 h. More preferably, the hydrolysis pH is 8.0 and the hydrolysis temperature is 45℃.

[0016] Preferably, the enzyme inactivation method described in S1 is enzyme inactivation at 95~100℃ for 10~12 min. A further preferred enzyme inactivation method is enzyme inactivation at 100℃ for 10 min.

[0017] Preferably, the volume concentration of ethanol after adding ethanol in S1 is 75% to 85%. More preferably, the volume concentration of ethanol is 80%.

[0018] Preferably, the chromatographic column for the gel chromatography in S2 is a 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 the preparative reversed-phase high-performance liquid chromatography described in S2 include: an Agilent Prep C18 column with dimensions of 100 Å, 5 µm, and 30 × 100 mm; a detection wavelength of 220 nm; a column temperature of 30 °C; mobile phase A being a 0.05%–0.1% trifluoroacetic acid (TFA) aqueous solution; mobile phase B being an acetonitrile aqueous solution containing 0.05%–0.1% trifluoroacetic acid, wherein the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 84%–85%; elution gradient with the following elution program: 0–50 min 96%–50% A; 50–54 min 50%–0% A; 54–60 min 0% A; and collection of the eluent after 8–12 min.

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

[0022] The third aspect of this invention provides the application 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 this invention provides the application 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 this invention provides the use of the above-mentioned pancreatic lipase inhibitory peptide or the pancreatic lipase inhibitory peptide prepared by the above-mentioned method in the preparation of a pharmaceutical excipient for the treatment and / or prevention of obesity, wherein the active ingredient comprises the above-mentioned pancreatic lipase inhibitory peptide or the pancreatic lipase inhibitory peptide prepared by the above-mentioned method. The pharmaceutical excipient can be used alone for the treatment and / or prevention of obesity, or it can be used in combination with other drugs for the treatment or prevention of obesity to enhance the therapeutic effect.

[0025] The sixth aspect of this invention provides the application of the above-described pancreatic lipase inhibitory peptide, or the pancreatic lipase inhibitory peptide prepared by the above-described method, in the preparation of a topical product for reducing subcutaneous fat, wherein the active ingredient includes the above-described pancreatic lipase inhibitory peptide or the pancreatic lipase inhibitory peptide prepared by the above-described method. The topical product includes slimming creams, etc.

[0026] The beneficial effects of this invention are as follows: (1) The amino acid sequences of the pancreatic lipase inhibitory peptides provided by the present invention are all derived from food proteins. The toxicity verification by Toxinpred showed that there was no risk of toxicity accumulation. The long-term safety of these peptides is significantly better than that of chemically synthesized inhibitors (such as orlistat), and they can effectively avoid the side effects such as intestinal discomfort that may be caused by chemically synthesized inhibitors. Furthermore, 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 being digested in the gastrointestinal tract. (2) Experiments have shown that the above-mentioned oligopeptides FGF, LLY, LYL, FAP and FWG have excellent inhibitory activity against pancreatic lipase, with half-inhibitory concentrations of 3.579 mmol / L, 6.308 mmol / L, 8.539 mmol / L, 9.278 mmol / L and 11.860 mmol / L, respectively, which can effectively limit the free fatty acids generated during lipid hydrolysis from entering the bloodstream to synthesize fat; (3) The oligopeptides in the pancreatic lipase inhibitory peptides provided by this invention are all tripeptides with simple structures, which can be directly prepared by artificial synthesis. Furthermore, the tripeptide structure allows them to specifically bind to the catalytic triplet (Ser152-Asp176-His263) or substrate-binding domain of pancreatic lipase, forming a non-competitive reversible inhibition. This non-competitive inhibition mechanism makes them unaffected by substrate concentration, and they can still effectively inhibit lipolysis in the complex intestinal environment. In addition, compared to other pancreatic lipase inhibitory peptides, the above-mentioned oligopeptides have smaller molecular weights, so they can be directly absorbed into the body by the intestines and exert their effects, resulting in higher utilization. (4) The above-mentioned oligopeptides have good water solubility and can be directly added to solid beverages (such as meal replacement powder), dairy products or nutrition bars without affecting the taste of 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 method for preparing pancreatic lipase inhibitory peptide provided by the present invention extracts the above-mentioned oligopeptides from sea cucumbers through enzymatic hydrolysis technology. The entire process from raw material screening to final product preparation follows the concept of green biomanufacturing, which is safer and has a lower cost than chemical synthesis route, and has economic feasibility for large-scale production. (6) This invention broadens the commercial application of sea cucumber polypeptides and is of great significance for developing safer health foods that help control body fat. Attached Figure Description

[0027] Figure 1 This is a high-performance liquid chromatogram of the oligopeptide FGF synthesized by solid-phase method in Example 4 of the present invention; Figure 2 This is the mass spectrum of the oligopeptide FGF synthesized by solid-phase method in Example 4 of the present invention; Figure 3 This is a high-performance liquid chromatogram of the oligopeptide LLY synthesized by solid-phase method in Example 5 of the present invention; Figure 4 This is the mass spectrum of the oligopeptide LLY synthesized by solid-phase method in Example 5 of the present invention; Figure 5 This is a high-performance liquid chromatogram of the oligopeptide LYL synthesized by solid-phase method in Example 6 of the present invention; Figure 6 This is the mass spectrum of the oligopeptide LYL synthesized by solid-phase method in Example 6 of the present invention; Figure 7 This is a high-performance liquid chromatogram of the oligopeptide FAP synthesized by solid-phase method in Example 7 of the present invention; Figure 8 This is the mass spectrum of the oligopeptide FAP synthesized by solid-phase method in Example 7 of the present invention; Figure 9This is a high-performance liquid chromatogram of the oligopeptide FWG synthesized by solid-phase method in Example 8 of the present invention; Figure 10 This is the mass spectrum of the oligopeptide FWG synthesized by solid-phase method in Example 8 of this invention; Figure 11 This is a global image and molecular interaction diagram of the molecular docking between oligopeptide FGF and pancreatic lipase in Example 1 of this invention. Figure 12 This is a global image and molecular interaction diagram of the molecular docking between oligopeptide LLY and pancreatic lipase in Example 1 of this invention. Figure 13 This is a global image and molecular interaction diagram of the molecular docking between oligopeptide LYL and pancreatic lipase in Example 1 of this invention. Figure 14 This is a global image and molecular interaction diagram of the molecular docking between oligopeptide FAP and pancreatic lipase in Example 1 of this invention; Figure 15 This is a global image and molecular interaction diagram of the molecular docking between oligopeptide FWG and pancreatic lipase in Example 1 of this invention. Figure 16 The IC50 of pancreatic lipase inhibitory activity in Test Example 2 of this invention is... 50 Value measurement results. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0029] Pancreatic lipase (PL) is a key enzyme in lipid hydrolysis and an effective target for obesity prevention. Effectively inhibiting PL activity holds promise for weight loss and prevention of obesity-related diseases. While drugs commonly used to inhibit PL activity are effective in controlling obesity, they also cause various side effects. Bioactive peptides from food sources are typically natural, safe, and readily available, and are frequently used in the development of both general and health foods. Sea cucumbers are high in protein and contain a complete range of amino acids, making them an excellent source of marine bioactive peptides. Studies have shown that sea cucumber peptides possess various bioactivities, including anti-fatigue and performance-enhancing effects, cardiovascular protection, antioxidant and anti-inflammatory properties, immunomodulatory and anti-tumor adjuvant effects, and wound healing promotion. However, sea cucumber peptides have not yet been found to inhibit pancreatic lipase activity.

[0030] This invention, through experimental research, obtained a pancreatic lipase inhibitory peptide from sea cucumber polypeptides. This pancreatic lipase inhibitory peptide contains at least one oligopeptide with the amino acid sequence of phenylalanine-glycine-phenylalanine, leucine-leucine-tyrosine, leucine-tyrosine-leucine, phenylalanine-alanine-proline, and phenylalanine-tryptophan-glycine. The above-mentioned oligopeptides exhibit excellent inhibitory activity against pancreatic lipase, and are all derived from food proteins, thus possessing high safety. Their small molecular weight makes them easier to absorb, and they can target and bind to the pancreatic lipase catalytic triplet or substrate-binding domain, forming a non-competitive reversible inhibition mode, thereby providing a long-lasting lipid-control advantage.

[0031] This invention also provides a method for preparing pancreatic lipase inhibitory peptides, specifically including the following steps: S1. Prepare sea cucumber homogenate by mixing sea cucumber and water at a ratio of 1:(18~20). First, hydrolyze the homogenate with pepsin, then hydrolyze it with Bacillus subtilis protease. After hydrolysis, inactivate the enzymes and add ethanol to the hydrolysate to remove sugar, thus obtaining sea cucumber protease hydrolysate. S2. The sea cucumber protein hydrolysate obtained in S1 is subjected to ultrafiltration using gel chromatography to collect components with a molecular weight less than 1 kDa. The obtained components are then separated and purified using preparative reverse high performance liquid chromatography to obtain the pancreatic lipase inhibitory peptide.

[0032] The present invention will be described below through specific embodiments.

[0033] The porcine pancreatic lipase (type II) used in the following examples was purchased from Sigma-Aldrich (catalog number 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 Biomedical Technology Co., Ltd. Oligopeptides were synthesized at Hefei Sener Biotechnology Co., Ltd.

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

[0035] The solutions used in the following examples are prepared by the following methods: 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 homogeneous to prepare a 10 mg / mL pancreatic lipase solution; take 1 mL of this solution 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-Nitrophenylbutyrate (p-NPB) solution: Weigh 0.0523 g of p-nitrophenylbutyrate and dilute to a 25 mL brown volumetric flask with anhydrous ethanol; Simulated gastric juice (SGF): 34 mM NaCl, 0.15% (w / v) pepsin, and 1 mol / L hydrochloric acid adjusted to pH 1.5-2.0; Simulated intestinal fluid (SIF): 50 mM KH2PO4 buffer, 10 mM sodium taurocholate (bile salt), 1.0 mg / mL pancreatic enzyme, and 1 mol / L NaOH adjusted to pH 7.0±0.2; Termination solution: 0.5 mM Pepstatin A (pepsin inhibitor), 1 mM PMSF (serine protease inhibitor).

[0036] Unless otherwise specified, the raw materials, reagents, pharmaceuticals, or instruments used in the following embodiments are all commercially available products. The methods used in the following embodiments are, unless otherwise specified, conventional methods in the art.

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

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

[0039] (2) Enzymatic hydrolysis: Sea cucumber was homogenized with water at a ratio of 1:20. Pepsin was selected to perform preliminary hydrolysis at 2000 U / g under acidic conditions. The hydrolysis parameters were pH 2.5, hydrolysis temperature 37℃, and hydrolysis time 2 h. Then, the pH was adjusted to 8.0 with 50 mM Tris-HCl solution. Bacillus subtilis protease was added at 6000 U / g and hydrolyzed at 45℃ for 3 h. The reaction was terminated by boiling water bath for 10 min.

[0040] (3) Sugar removal: Centrifuge the enzymatic hydrolysate obtained in step (1) at a speed of 8000 r / min for 15 min. Then take the supernatant and evaporate it by rotary evaporation. After concentrating it to 1 / 5 of the original volume, slowly add four times the volume of anhydrous ethanol. Mix well and let it stand at 4℃ for 72 h. Centrifuge again and evaporate the supernatant by rotary evaporation to concentrate it. Freeze dry to obtain sea cucumber enzymatic hydrolysate.

[0041] (4) Gel chromatography separation: The sea cucumber protein hydrolysate obtained in step (3) was dissolved in ultrapure water, centrifuged (10000×g, 20 min) to remove insoluble particles, and the supernatant was used to separate small molecule peptides using a 3 kDa ultrafiltration tube. Gel chromatography was used for separation and purification, and the eluent with a molecular weight less than 1 kDa was collected. The gel chromatography column was a TK-Col TA-GF30 (1.6 cm × 60 cm), the UV detector wavelength was 220 nm, the equilibration buffer was ultrapure water, the injection concentration was 10 mg / mL, and the injection volume was 1 mL.

[0042] (5) Preparative reversed-phase high-performance liquid chromatography (RP-HPLC): The eluent obtained in step (4) was separated and purified using preparative reversed-phase high-performance liquid chromatography to obtain high-purity sea cucumber oligopeptides. The chromatographic column used in the preparative reversed-phase high-performance liquid chromatography was an Agilent Prep C18 (100 Å, 5 µm, 30 × 100 mm), the detection wavelength was 220 nm, the column temperature was 30 °C, mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA acetonitrile aqueous solution (acetonitrile concentration was 84%). The elution gradient was as follows: 0 ~ 50 min 96%-50% A; 50 ~ 54 min 50% ~ 0% A; 54 ~ 60 min 0% A; the eluent was collected after 8 ~ 12 min.

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

[0044] (7) Inhibition rate determination: The sea cucumber oligopeptide powder obtained in step (6) was prepared into a polypeptide solution with DMSO (<1%) solution and the inhibition rate of pancreatic lipase activity was determined (the method is the same as in test example 2). The solution concentration was 15 mg / mL and the inhibition rate was 40.12%.

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

[0046] The enzymatic hydrolysis products were separated by capillary high-performance liquid chromatography (HPLC) and then analyzed by mass spectrometry using a Q Exactive HF-X mass spectrometer (ThermoFisher) for 60 min. Detection mode: positive ion. The mass-charge ratio of peptides and peptide fragments was collected using the following method: 10 fragment spectra were acquired after each full scan (MS2 scan), resulting in 1403 peptide fragments, including oligopeptides FGF, LLY, LYL, FAP, and FWG.

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

[0048] (1) Raw material pretreatment: Select fresh sea cucumbers as raw materials, remove internal organs and surface dirt and salt, and repeatedly soak and change water to reduce impurity content.

[0049] (2) Enzymatic hydrolysis: Sea cucumber was homogenized with water at a ratio of 1:20. Pepsin was selected to perform preliminary hydrolysis at 1500 U / g under acidic conditions. The hydrolysis parameters were pH 2.4, hydrolysis temperature 35℃, and hydrolysis time 2 h. Then, the pH was adjusted to 7.9 with 50 mM Tris-HCl solution. Bacillus subtilis protease was added at 4000 U / g and hydrolyzed at 43℃ for 3.5 h. The reaction was terminated by boiling water bath for 10 min.

[0050] (3) Sugar removal: Same as in Example 1.

[0051] (4) Gel chromatography separation: The sea cucumber protein hydrolysate obtained in step (3) was dissolved in ultrapure water, centrifuged (8000×g, 20 min) to remove insoluble particles, and the supernatant was used to separate small molecule peptides using a 3 kDa ultrafiltration tube. Gel chromatography was used for separation and purification, and the eluent with a molecular weight less than 1 kDa was collected. The gel chromatography column was a TK-Col TA-GF30 (1.6 cm × 60 cm), the UV detector wavelength was 220 nm, the equilibration buffer was ultrapure water, the injection concentration was 10 mg / mL, and the injection volume was 1 mL.

[0052] (5) Preparative reversed-phase high-performance liquid chromatography (RP-HPLC): The eluent obtained in step (4) was separated and purified using preparative reversed-phase high-performance liquid chromatography to obtain high-purity sea cucumber oligopeptides. The preparative HPLC column was an Agilent Prep C18 (100 Å, 5 µm, 30 × 100 mm), the detection wavelength was 220 nm, the column 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 85%). The elution gradient was as follows: 0 ~ 50 min 96%-50% A; 50 ~ 54 min 50% ~ 0% A; 54 ~ 60 min 0% A; the eluent was collected after 8 ~ 12 min.

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

[0054] (7) Separation of active peptides: The sea cucumber oligopeptide powder obtained in step (6) is separated by chromatography under the same chromatographic conditions as in Example 1. The eluent is collected according to the elution time of FGF, LLY, LYL, FAP and FWG to obtain high-purity FGF, LLY, LYL, FAP and FWG.

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

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

[0057] (2) Enzymatic hydrolysis: Sea cucumber was homogenized with water at a ratio of 1:20. Pepsin was selected to perform preliminary hydrolysis at 2500 U / g under acidic conditions. The hydrolysis parameters were pH 2.6, hydrolysis temperature 39℃, and hydrolysis time 1 h. Then, the pH was adjusted to 8.1 with 50 mM Tris-HCl solution. Bacillus subtilis protease was added at 8000 U / g and hydrolyzed at 47℃ for 3 h. The reaction was terminated by maintaining the solution in a 95℃ water bath for 12 min.

[0058] (3) Sugar removal: Same as in Example 1.

[0059] (4) Gel chromatography separation: Same as in Example 1.

[0060] (5) Preparative reversed-phase high-performance liquid chromatography (RP-HPLC): The eluent obtained in step (4) was separated and purified using preparative reversed-phase high-performance liquid chromatography to obtain high-purity sea cucumber oligopeptides. The chromatographic column used in the preparative reversed-phase high-performance liquid chromatography was an Agilent Prep C18 (100 Å, 5 µm, 30 × 100 mm), the detection wavelength was 220 nm, the column 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 was as follows: 0 ~ 50 min 96%-50% A; 50 ~ 54 min 50% ~ 0% A; 54 ~ 60 min 0% A; the eluent was collected after 8 ~ 12 min.

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

[0062] (7) Separation of active peptides: The sea cucumber oligopeptide powder obtained in step (6) is separated by chromatography under the same chromatographic conditions as in Example 1. The eluent is collected according to the elution time of FGF, LLY, LYL, FAP and FWG to obtain high-purity FGF, LLY, LYL, FAP and FWG.

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

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

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

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

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

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

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

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

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

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

[0073] Example 9 This embodiment provides a pancreatic lipase inhibitory peptide, which is a compound of the oligopeptides of Examples 4-8 in a mass ratio of 1:1:1:1:1.

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

[0075] According to ToxinPred's predictions, oligopeptides FGF, LLY, LYL, FAP, and FWG are non-toxic and non-carcinogenic.

[0076] Based on gastrointestinal absorption capacity and the Lipinski five principles, oligopeptides FGF, LLY, LYL, FAP, and FWG are predicted to have good gastrointestinal absorption capacity and high potential bioactivity.

[0077] The 3D structure of pancreatic lipase (PDB ID: 1LPB) was downloaded from the RCSB pdb database. Structural preprocessing was performed using Pymol, including adding hydrogen atoms, atomic dehybridization, removing water molecules, and removing existing ligands from the protein molecule. 3D models of oligopeptides FGF, LLY, LYL, FAP, and FWG were constructed using Pymol. Molecular docking was performed using AutoDock Vina 1.1.2.0 (the Scripps Research Institute, La Jolla, CA, USA), and visualization was performed using Pymol. The docking engine was AutoDock Vina, the docking box size was set to 18.0 × 19.3 × 16.6 ų, and the center point coordinates were (x, y, z) = (4.1, 27.7, 48.7).

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

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

[0080] Table 1. Binding energy of active peptides to pancreatic lipase inhibition

[0081] Test Example 2 This test case investigated the half-maximal inhibitory concentrations (WMCs) of the oligopeptides FGF, LLY, LYL, FAP, and FWG from Examples 4 to 8 on pancreatic lipase.

[0082] The in vitro inhibitory activity of porcine pancreatic lipase (type II) was determined using p-nitrophenylbutyrate (p-NPB) as a substrate.

[0083] Sample experimental group: 50 µL of oligopeptide solution (concentration of 100 µM ~ 10 mM, solvent of 0.1% DMSO) and 50 µL of 0.25 mg / mL pancreatic lipase solution were mixed in a 96-well microplate and incubated at 25°C for 15 min. Then, 50 µL of p-NPB solution was added and reacted in the dark for 10 min before being measured by an enzyme-linked immunosorbent assay (ELISA) reader.

[0084] Control group: 50 µL of 0.25 mg / mL pancreatic lipase solution was placed in a 96-well microplate and incubated at 25°C for 15 min. Then, 50 µL of p-NPB solution was added and reacted in the dark for 10 min before being measured by an enzyme-linked immunosorbent assay (ELISA) reader.

[0085] Sample blank group: 50 µL of oligopeptide solution (concentration of 100 µM to 10 mM, solvent of 0.1% DMSO) was placed in a 96-well microplate and incubated at 25°C for 15 min. Then, 50 µL of p-NPB solution was added and reacted in the dark for 10 min before being measured by an ELISA reader.

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

[0087] The inhibition rate of pancreatic lipase activity is calculated using 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] The inhibition rate of pancreatic lipase activity of the positive control orlistat was determined using the same method.

[0091] The results of the fitting analysis and significance analysis using Graphpad Prism 9 and SPSS software are shown in Table 2. Figure 16 As shown, the half-maximal inhibitory concentration (IC50) of the positive control orlistat is... 50The concentration of the oligopeptide was 0.976 mmol / L, and the IC50 values ​​for oligopeptides FGF, LLY, LYL, FAP, and FWG were [not specified]. 50 The values ​​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. IC50 of active peptides on pancreatic lipase inhibition 50 value

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

[0094] Test Example 3 This test case investigated the bioactivity of the oligopeptides FGF, LLY, LYL, FAP, and FWG from Examples 4-8, as well as the positive control orlistat, in the gastrointestinal tract.

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

[0096] Add 1 mL of simulated gastric fluid (SGF), mix well, and bring the final volume to 2 mL. Incubate at 37°C with shaking 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 products and mix well. Incubate at 37°C with shaking for 2 h, then add 10 μL of 1 mM PMSF and mix well to obtain the digestion termination solution.

[0098] 3. Undigested group: Oligopeptide samples were not treated with SGF / SIF and were directly subjected to subsequent activity assays.

[0099] 4. Sample processing after digestion Transfer the digestion stop solution to an ultrafiltration centrifuge tube (3 kDa MWCO). Centrifuge at 12,000 × g, 4 °C for 30 min, and collect the filtrate. Dilute the filtrate to a suitable concentration with 50 mM Tris-HCl solution for activity assay.

[0100] 5. Inhibition rate calculation The inhibition rate of pancreatic lipase activity was determined using the method described in Example 2, and the IC50 values ​​before and after digestion were calculated. 50 And compare the activity retention rate.

[0101] Activity retention rate (%) = (IC50 of undigested group) 50 / Post-digestion IC 50 ) × 100%.

[0102] The IC50 of the positive control orlistat before and after digestion was determined using the same method. 50 and activity retention rate.

[0103] 6. Results As shown in Table 3, oligopeptides FGF, LLY, LYL, FAP, and FWG retain more than 80% of their activity after being digested in the gastrointestinal tract.

[0104] Table 3 IC before and after digestion 50 and activity retention rate

[0105] Note: a, b, c, d, and e represent significant differences between different samples.

[0106] 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, and improvements 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 sea cucumber oligopeptide powder, characterized in that, Specifically, the following steps are included: S1. Prepare a sea cucumber homogenate by mixing sea cucumber and water at a material-to-liquid ratio of 1:(18~20). First, hydrolyze the homogenate with pepsin, then with Bacillus subtilis protease. After hydrolysis, inactivate the enzymes and add ethanol to the resulting hydrolysate to remove sugar, thus obtaining sea cucumber protease hydrolysate. The amount of pepsin added for hydrolysis is 1500~2500 U / g dried sea cucumber, the hydrolysis pH is 2.4~2.6, the hydrolysis temperature is 35~39℃, and the hydrolysis time is 1~2 h. The amount of Bacillus subtilis protease added for hydrolysis is 4000~8000 U / g dried sea cucumber, the hydrolysis pH is 7.9~8.1, the hydrolysis temperature is 43~47℃, and the hydrolysis time is 3~3.5 h. The volume concentration of the ethanol after adding ethanol is 75%~85%. S2. The sea cucumber protein hydrolysate obtained in S1 was subjected to ultrafiltration using gel chromatography to collect components with a molecular weight less than 1 kDa. The obtained components were then separated and purified using preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain the sea cucumber oligopeptide powder. The gel chromatography column was a TK-Col TA-GF30, the UV detector wavelength was 220 nm, and the equilibration buffer was ultrapure water. The chromatographic conditions for the preparative reversed-phase HPLC included: an Agilent Prep C18 column (100 Å, 5 µm, 30 × 100 mm), a detection wavelength of 220 nm, a column temperature of 30 °C, mobile phase A being a 0.05%–0.1% trifluoroacetic acid aqueous solution, and mobile phase B being an acetonitrile aqueous solution containing 0.05%–0.1% trifluoroacetic acid, with an acetonitrile volume percentage of 84%–85%; the elution gradient program was 0–50 min 96%–50% A; 50–54 min 50% A. ~0%A; 54~60min 0%A; collect the effluent after 8~12 min; The sea cucumber oligopeptide powder contains phenylalanine-glycine-phenylalanine, leucine-leucine-tyrosine, leucine-tyrosine-leucine, phenylalanine-alanine-proline, and phenylalanine-tryptophan-glycine.

2. The preparation method according to claim 1, characterized in that, The feed-to-liquid ratio described in S1 is 1:20; and / or The enzyme inactivation method described in S1 is to inactivate the enzyme at 95~100℃ for 10~12 min.

3. The preparation method according to claim 2, characterized in that, S1 is hydrolyzed with pepsin at a pH of 2.5 and a hydrolysis temperature of 37°C; and / or In S1, the enzymatic hydrolysis using Bacillus subtilis protease occurred at a pH of 8.0 and a hydrolysis temperature of 45°C; and / or The volume concentration of ethanol after adding ethanol to S1 is 80%.

4. The preparation method according to claim 1, characterized in that, Mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, and mobile phase B is an acetonitrile aqueous solution containing 0.1% trifluoroacetic acid, wherein the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 84%.

5. The application of sea cucumber oligopeptide powder prepared by the preparation method according to any one of claims 1 to 4 in the preparation of health food that helps control body fat.

6. The use of sea cucumber oligopeptide powder prepared by the preparation method according to any one of claims 1 to 4 in the preparation of pharmaceutical excipients for the treatment and / or prevention of obesity, characterized in that, The active ingredient of the pharmaceutical excipient includes sea cucumber oligopeptide powder prepared by the preparation method according to any one of claims 1 to 4.

7. The application of the sea cucumber oligopeptide powder prepared by the preparation method according to any one of claims 1 to 4 in the preparation of topical products for reducing subcutaneous fat, characterized in that, The active ingredient of the topical product includes sea cucumber oligopeptide powder prepared by the preparation method according to any one of claims 1 to 4.

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