Horse milk-derived small molecule peptide and application thereof

Abstract

The invention discloses a horse milk-derived small molecule peptide, the amino acid sequence of which is Phe-Gly-Gly-Leu-Met (FGGLM), and the molecular weight of which is 523.64 Da. The small molecule peptide disclosed by the invention has the activity of inhibiting pancreatic lipase (PL), the inhibition rate of the pancreatic lipase is 75.95%, the accumulation of the content of triglyceride (TG) in oleic acid-induced human liver cancer (Hepg2) cells can be reduced, and the human liver cancer (Hepg2) cells can be inhibited. The small molecule peptide can be applied to preparation of preparations for treating or assisting in treating hyperlipidemia and obese people, and has the characteristics of simplicity in preparation and suitability for industrial production and market popularization and application.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a horse milk-derived small molecule peptide and application thereof. Background Art

[0002] Pancreatic lipase is a type of enzyme with lipolytic activity, which plays an important role in lipid metabolism. The inhibition and regulation of pancreatic lipase is an ideal target for the prevention and treatment of metabolic diseases such as hyperlipidemia and obesity. Common lipid-lowering drugs in clinical practice include statins, fibrates, and cholesterol absorption inhibitors. Although these drugs can effectively regulate dyslipidemia and lower plasma cholesterol, they also have many side effects such as rhabdomyolysis and gastrointestinal allergic reactions.

[0003] Bioactive peptides have a variety of human metabolic and physiological regulatory functions. A variety of lipid-lowering peptides have been isolated from the hydrolyzates of protein sources such as whey, soybeans, and chlorella. The earliest sequence identification was from milk protein peptides. Therefore, the development of natural lipid-lowering active substances with abundant sources, safety and effectiveness has become a key research direction. Summary of the invention

[0004] The invention provides a small molecule peptide derived from fermented mare's milk, the amino acid sequence of which is Phe-Gly-Gly-Leu-Met (FGGLM), the molecular weight of which is 523.64 Da, the small molecule peptide has pancreatic lipase inhibitory activity, and can be used for preparing a pancreatic lipase inhibitor.

[0005] Another object of the present invention is to use the fermented horse milk-derived small molecule peptide in the preparation of an anti-obesity or hyperlipidemia prevention and treatment preparation.

[0006] The ingredients (or active ingredients) of the preparation of the present invention are the above-mentioned small molecule peptides, and one or more drug- or food-acceptable excipients may be added to improve the absorption effect of the drug or food or facilitate use, and to prepare suitable dosage forms, such as capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, suitable dosage forms for use in pharmacy, or suitable ways of consumption in the food field; it can be used to prepare functional foods, health products and drugs for treating or assisting in the treatment of obesity or preventing and treating hyperlipidemia.

[0007] The object of the present invention is achieved through the following scheme:

[0008] 1. After sterilizing at 65-115° C. for 2-15 min, fresh mare's milk is inoculated with lactic acid bacteria and yeast, the inoculation amount is 0.1-10%, the fermentation temperature is 20-37° C., the shaking speed is 50-300 rpm, and the fermentation time is 16-90 h; the lactic acid bacteria is Lactobacillus paracasei; the yeast is Dekkera heteromorpha and Kazakhstan monosporic yeast;

[0009] 2. After the fermentation of fresh mare's milk is completed, 0.3% (w / v) pepsin is added, and the pH of the fermented mare's milk is adjusted to 4.0-4.8 with 5M HCl, and the mixture is treated at 30-38°C and 50-100rpm for 2-4h; then the pH value is adjusted to 7.0 with 5M NaOH, 0.1% (w / v) trypsin is added, and the mixture is treated at 37°C and 60rpm for 2-4h, and the supernatant is taken by centrifugation at 4°C and 12000g for 10-15min, and freeze-dried to obtain freeze-dried powder;

[0010] 3. The lyophilized powder was dissolved in distilled water to prepare a solution of 0.05-5 g / mL, and filtered with an ultrafiltration membrane with a cutoff of 10 kDa and 3 kDa to obtain a solution containing components with molecular weights>10 kDa, 3-10 kDa, and <3 kDa, and freeze-dried respectively. The lyophilized powders were collected, and the lyophilized powders containing different components were dissolved in distilled water respectively, and the pancreatic lipase inhibitory activity of each component was detected. The liquid with the best inhibitory activity was selected for separation and purification by dextran gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and the peak with pancreatic lipase inhibitory activity after RP-HPLC purification was selected for LC-MS identification. The peptide sequence obtained after identification was predicted for activity in BIOPEP, and the α-fold3 and CB-Dock2 databases were used for molecular docking simulation and pancreatic lipase inhibitory activity prediction. The one with the best predicted activity was selected for solid phase synthesis verification, and finally the small molecule peptide of the present invention was obtained.

[0011] The fermented horse milk-derived small molecule peptide Phe-Gly-Gly-Leu-Met of the present invention can significantly inhibit the activity of pancreatic lipase, improve the accumulation of triglyceride content in Hepg2 cells induced by oleic acid, and reduce the accumulation of lipid droplets in Hepg2 cells induced by oleic acid; the small molecule peptide of the present invention can be used to prepare a medicament or food for alleviating or assisting in the treatment of obesity or hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of the inhibitory activity of the solution containing substances of different molecular weights on pancreatic lipase after ultrafiltration retentate;

[0013] Figure 2 Schematic diagram of the separation peaks after separation of components with molecular weight <3 kDa by RP-HPLC;

[0014] Figure 3 The results of the pancreatic lipase inhibitory activity test of the separation solution corresponding to the separation peak after RP-HPLC separation;

[0015] Figure 4 The RP-HPLC separation results of the separation solution in the 35-40 min period;

[0016] Figure 5 is the high performance liquid chromatogram of the separation liquid of peak 2;

[0017] Figure 6 This is the mass spectrum identification diagram of the separation solution of peak 2;

[0018] Figure 7 The schematic diagram of molecular docking between small molecule peptide FGGLM and pancreatic lipase;

[0019] Figure 8 The results of the in vitro inhibition test of pancreatic lipase activity by the small molecule peptide FGGLM;

[0020] Fig. 9 The results of the effect of small molecule peptide FGGLM on the activity of Hepg2 cells;

[0021] Fig.10 This is the effect of the small molecule peptide FGGLM on the accumulation of triglyceride content in Hepg2 cells induced by oleic acid. DETAILED DESCRIPTION

[0022] The present invention is further described below by way of examples, but the protection of the present invention is not limited to the described contents. The methods in the present embodiments are all performed according to conventional methods unless otherwise specified, and the experimental equipment, reagents, etc. used are all obtained from commercial channels unless otherwise specified.

[0023] In the following examples, the pancreatic lipase solution was prepared with Tris-HCl buffer, the small molecule peptide solution was prepared with deionized water, and the p-NPB substrate solution was prepared with DMSO;

[0024] After the pancreatic lipase solution and the small molecule peptide solution were mixed, they were preheated at 37°C for 10 min, and then the p-NPB substrate solution was added at 37°C for 15-30 min. The reaction product was centrifuged at 5000 g for 5 min, and the supernatant was taken and its absorbance value was detected at 405 nm (experimental group);

[0025] At the same time, set up the experimental blank group: Tris-HCl buffer + small molecule peptide + p-NPB;

[0026] Control group: Tris-HCl buffer + PL + pN Tris-HCl buffer PB, without small molecule peptide;

[0027] Control blank group: Tris-HCl buffer + p-NPB, without small molecule peptides and pancreatic lipase;

[0028] Orlistat was added as a positive control;

[0029] The reaction system was prepared according to Table 1. The experiment was repeated three times, and the inhibition rate was calculated according to the following formula:

[0030] Table 1 Reaction system for pancreatic lipase activity determination

[0031]

[0032]

[0033] Experimental Example 1: Acquisition of small molecule peptides

[0034] (1) fresh mare's milk was sterilized at 115° C. for 10 min; three generations of activated Dekkera heteromorpha, Kazakhstan monosporus and Lactobacillus paracasei were inoculated into the sterilized fresh mare's milk at an inoculation ratio of 2% for yeast and 4% for lactic acid bacteria, and then fermented at 37° C. and 150 rpm for 48 h;

[0035] (2) adding 0.3% (w / v) pepsin to the fermented mare's milk solution, adjusting the pH of the fermented mare's milk to 4.30 with 5M HCl, and treating at 37°C and 60 rpm for 2 h; then adjusting the pH to 7.0 with 5M NaOH, adding 0.1% (w / v) trypsin, treating at 37°C and 60 rpm for 2 h, and then centrifuging at 4°C and 12000g for 15 min, taking the supernatant, and freeze-drying to obtain a lyophilized powder;

[0036] (3) The lyophilized powder was dissolved in deionized water to prepare a 100 mg / mL solution. The component was filtered using two ultrafiltration membranes with different cutoffs of 10 kDa and 3 kDa to obtain three different components of >10 kDa, 3-10 kDa, and <3 kDa. The three components were lyophilized using a vacuum freeze dryer and further prepared into a 50 mg / mL solution using deionized water. The inhibition rate of each component on pancreatic lipase activity was determined. The results are shown in FIG. Figure 1 As shown in the figure, the component solution with a molecular weight of <3 kDa has the best inhibitory effect on pancreatic lipase activity. The component solution was further separated by RP-HPLC. The results are as follows Figure 2 As shown, according to different time periods Figure 2 The peaks in the sample were divided into 7 parts. The separated solutions at different time periods were collected and freeze-dried after removing the mobile phase using a rotary evaporator. The freeze-dried components at each time period were prepared into 10 mg / mL solutions with deionized water. The inhibitory activity of the solutions of different components on pancreatic lipase was determined. The results are shown in Figure 3 As shown in the figure, the component with the best inhibitory effect on pancreatic lipase activity is the separated liquid in the 35-40min period. The separated liquid of this component is further separated by RP-HPLC. The results are shown in Figure 4 The separated solutions of different peaks were collected and the mobile phase was removed by rotary evaporator and then freeze-dried. The inhibitory activity of the solutions of different peaks on pancreatic lipase was determined. The results showed that the separated solution of peak 2 had the best inhibitory effect on pancreatic lipase activity. The HPLC chromatogram of this peak is shown in Figure 5 ,from Figure 5It can be seen that the liquid phase results show a single peak, indicating that the separation is complete.

[0037] The chromatographic conditions of RP-HPLC are as follows: injection volume 1000 μL, flow rate 2 mL / min, detection wavelength at 215 nm; mobile phase A contains deionized water containing 0.1% (v / v) trifluoroacetic acid (TFA), mobile phase B is acetonitrile (CH3CN or C2H3N) containing 0.1% (v / v) TFA, and mobile phases A and B are used for gradient elution, and the elution program is: 0-5 min, 10% B; 5-40 min, 50% B; 40-50 min, 60% B; 50-60 min, 10% B; 60-70 min, 10% B.

[0038] Example 2: LC-MS identification

[0039] The peak component No. 2 was identified by LC-MS, and the analysis time was 30 minutes. Detection method: positive ion. The mass-to-charge ratio of small molecule peptides and small molecule peptide fragments was collected according to the following method: 10 fragment spectra (MS2 scan) were collected for each full scan (full scan); the mass spectrometry test raw file (Raw File) was searched against the corresponding database (Equuscaballus) using the software Mascot2.2, and finally the protein identification results were obtained.

[0040] See Figure 6 From the figure, we can get the corresponding intensity and charge-to-mass ratio of the corresponding fragments. Through sequence comparison with the database, it is confirmed that the small molecule peptide is FGGLM.

[0041] Experimental Example 3: Docking of small molecule peptide FGGLM with pancreatic lipase molecule

[0042] The three-dimensional model of pancreatic lipase (PL) was downloaded from the RCBSPDB protein database, and the three-dimensional structure of the small molecule peptide was drawn using α-fold3. The NM2 force field was added to minimize its energy. Gold 5.3.0 was used for flexible molecular docking, ligand extraction of receptors was performed, water molecules were removed, polar hydrogen was added, and the docking model was screened using the Gold score scoring system. The visualization analysis of the docked molecular complex was performed using Moe 2019.10 software. The results are shown in Figure 7 ,As can be seen from the figure, FGGLM forms two hydrogen bonds S153 and F78, and five hydrophobic bonds F216, P181, V260, I79 and W253 with PL, and the total binding energy is -8.1 kcal / mol.

[0043] Example 4: Detection of the inhibitory activity of small molecule peptide FGGLM on pancreatic lipase

[0044] The small molecule peptide sequence was synthesized by Shanghai Biotechnology Co., Ltd. with a purity of ≥98%. The obtained small molecule peptide was dissolved in a gradient manner to obtain 1, 2, 3, 4, and 5 mg / mL small molecule peptide solutions, and the inhibition of pancreatic lipase activity by small molecule peptide solutions of different concentrations was detected; the results are shown in Figure 8 The inhibition rate of small molecule peptide FGGLM on pancreatic lipase was 75.95% at a concentration of 5 mg / mL.

[0045] Experimental Example 5: Functional evaluation of small molecule peptide FGGLM

[0046] (1) Establishment of a high-lipid model of HepG2 cells induced by oleic acid (OA)

[0047] HepG2 cells were revived with DMEM high-glucose medium supplemented with 20% FBS and 1% penicillin-streptomycin; cultured in a 37°C, 5% CO2 incubator until the confluence reached 80%, digested with 0.25% trypsin, passaged at a ratio of 1:3, and the medium was replaced every 24 hours; after HepG2 cells were plated, the original culture supernatant was discarded, and a high-fat model was induced with DMEM medium containing 0.5 mmol / L oleic acid;

[0048] The model group (DC) cultured HepG2 cells with DMEM medium containing 0.5 mmol / L oleic acid;

[0049] The negative control group (NC) cultured HepG2 cells with DMEM medium without 0.5 mmol / L oleic acid;

[0050] The small molecule peptide group cultured HepG2 cells with different concentrations of small molecule peptides (1 mg, 2 mg, 3 mg, 4 mg, and 5 mg) and DMEM medium containing 0.5 mmol / L oleic acid;

[0051] The positive control group (PC) used DMEM medium containing 0.5mmol / L oleic acid and 25μmol / L orlistat to culture HepG2 cells;

[0052] For all groups, subsequent experiments were performed on cells after 24 h of culture;

[0053] (2) CCK-8 assay to detect the effect of FGGLM small molecule peptide on cell activity

[0054] HepG2 cells that had been cultured and plated to a rate of 80% were digested with trypsin and made into a cell suspension using DMEM medium containing 10% FBS. 5The cells were added to a 96-well plate, and then small molecule peptide solutions with concentrations of 1-5 mg / mL were added. At the same time, a control (NT) without small molecule peptide was set up. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h. Then, 10 μL CCK-8 reagent was added to each well of each group and cultured in a cell culture incubator for 4 h. The OD 450 The absorbance value was detected at 100 μl, and 3 replicates were performed in each group. The results are shown in Fig. 9 ,Depend on Fig. 9 It can be seen that the small molecule peptide FGGLM has no toxic side effects on HepG2 cells;

[0055] (3) Effect of small molecule peptide FGGLM on triglyceride accumulation in HepG2 cells

[0056] HepG2 cells that had been cultured and plated to a rate of 80% were digested with trypsin and made into a cell suspension using DMEM medium containing 10% FBS. 5 The cells were added to a 96-well plate and grouped according to step (1). The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h. The original culture medium was discarded, the cells were washed twice with PBS, and then trypsin was added to digest the cells in each group and centrifuged at 4°C and 3000 rpm for 5 min. 200 μL of cell lysis buffer was added to each group and lysed on ice for 30 min. After lysis, the cells were centrifuged at 4°C and 14000 rpm for 15 min to obtain the supernatant. The triglyceride content was determined according to the steps of the triglyceride determination kit of Nanjing Jiancheng Biological Company. The protein concentration of the precipitate was determined using the Biyuntian BCA protein determination kit. Three replicates were performed for each group. The results are shown in Table 1. Fig.10 As can be seen from the figure, the small molecule peptide FGGLM can significantly improve the accumulation of triglyceride content in high-fat HepG2 cells induced by oleic acid.

Claims

1. A horse milk-derived small molecule peptide, whose amino acid sequence is Phe-Gly-Gly-Leu-Met.

2. Use of the horse milk-derived small molecule peptide according to claim 1 in the preparation of an anti-obesity or hyperlipidemia prevention and treatment preparation.

3. The use according to claim 2, characterized in that :Small molecule peptides from horse milk have pancreatic lipase inhibitory activity.

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