Fermented horse milk source small molecule peptide and application thereof

Abstract

The invention discloses a small molecule peptide derived from fermented horse milk, the amino acid sequence of the small molecule peptide is His-Ala-Trp-Phe, and the molecular weight of the small molecule peptide is 559.67 Da. The small molecule peptide has pancreatic lipase inhibitory activity, can be applied to preparation of special medical foods and medicines for reducing hyperlipidemia, and is simple to prepare and suitable for industrial production and market popularization and application.

Description

Technical Field

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

[0002] Excessive fat intake can cause obesity and even hyperlipidemia, and the fat in food mainly exists in the form of triglycerides. Pancreatic lipase is the main lipase in the human body and plays an important role in the digestion of triglycerides. Dietary triglycerides are first hydrolyzed by pancreatic lipase, decomposed into chylomicrons, and then form micelles with cholesterol, bile salts, etc., and finally absorbed by cells in the intestine. Therefore, inhibiting pancreatic lipase can reduce the absorption of dietary fat and reduce diseases such as hyperlipidemia and obesity caused by a high-fat diet. At present, there are widely recognized pancreatic lipase inhibitor drugs on the market, such as orlistat. However, this drug has side effects such as insomnia, fatigue, nausea, vomiting, and flatulence. Therefore, it is necessary to find more effective and safer pancreatic lipase inhibitors from natural products, especially to obtain peptides with pancreatic lipase inhibitors from food proteins as alternative drugs for intervention in obesity, which has a huge potential market. Summary of the invention

[0003] The invention provides a fermented horse milk-derived small-molecule peptide, the amino acid sequence of which is His-Ala-Trp-Phe (HAWF) and the molecular weight is 559.67 Da. The small-molecule peptide has pancreatic lipase inhibitory activity.

[0004] 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.

[0005] 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, 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 hyperlipidemia.

[0006] The object of the present invention is achieved through the following scheme:

[0007] 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;

[0008] 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;

[0009] 3. Dissolve the lyophilized powder in double distilled water to prepare a solution with a concentration of 0.05-5 g / mL. Filter the solution using an ultrafiltration membrane with different molecular weight cutoffs to separate different fractions of liquid, and determine the pancreatic lipase inhibitory activity of each fraction. Select the ultrafiltration fraction with the strongest pancreatic lipase inhibitory activity for reverse phase high performance liquid chromatography (RP-HPLC) separation and purification. Liquid chromatography-mass spectrometry (LC-MS) was performed to identify the peak with pancreatic lipase inhibitory activity after RP-HPLC purification. The obtained peptide sequence was predicted for peptide activity using the BIOWARE database, and the α-fold3 and CB-Dock2 databases were used for molecular docking simulation and pancreatic lipase inhibitory activity prediction. The peptide sequence with the best predicted activity was selected for solid phase synthesis verification, and finally a new molecular peptide with pancreatic lipase inhibitory activity was obtained.

[0010] The fermented horse milk-derived small molecule peptide HAWF of the present invention can significantly inhibit the activity of pancreatic lipase, can be used to prepare special medical foods, health products and medicines for treating or assisting in the treatment of hyperlipidemia, and is simple to prepare and suitable for industrial production and market promotion applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The results of the inhibitory activity of the solution containing substances of different molecular weights on pancreatic lipase after ultrafiltration retentate;

[0012] Figure 2 Schematic diagram of the separation peaks after separation of components with molecular weight <3 kDa by RP-HPLC;

[0013] Figure 3 The results of the pancreatic lipase inhibitory activity test of the separation solution corresponding to the separation peak after RP-HPLC separation;

[0014] Figure 4 The RP-HPLC separation results of the separation solution in the 35-40 min period;

[0015] Figure 5 is the high performance liquid chromatogram of the separation liquid of peak 1;

[0016] Figure 6 This is the mass spectrum identification diagram of the separation solution of peak 1;

[0017] Figure 7 Schematic diagram of molecular docking between small molecule peptide HAWF and pancreatic lipase;

[0018] Figure 8 These are the results of the in vitro inhibition of pancreatic lipase activity by the small molecule peptide HAWF. DETAILED DESCRIPTION

[0019] The technical scheme of the present invention is further described in detail below through examples, but the content of the present invention is not limited thereto. The methods in the present examples are conventional methods unless otherwise specified, and the materials and reagents used are obtained from commercial sources unless otherwise specified;

[0020] 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;

[0021] 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);

[0022] At the same time, set up the experimental blank group: Tris-HCl buffer + small molecule peptide + p-NPB;

[0023] Control group: Tris-HCl buffer + PL + pN Tris-HCl buffer PB, without small molecule peptide;

[0024] Control blank group: Tris-HCl buffer + p-NPB, without small molecule peptides and pancreatic lipase;

[0025] Orlistat was added as a positive control;

[0026] 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:

[0027] Table 1 Reaction system for pancreatic lipase activity determination

[0028]

[0029]

[0030] Example 1: Acquisition of small molecule peptides

[0031] 1. Sterilize fresh mare's milk at 115°C for 10 min; inoculate the three-generation activated yeast, Kazakhstan monosporic yeast and Lactobacillus paracasei into the sterilized fresh mare's milk at an inoculation ratio of 2% for yeast and 4% for lactic acid bacteria, and then ferment at 37°C and 150rpm for 48h;

[0032] 2. Add 0.3% (w / v) pepsin to the fermented mare's milk solution, adjust the pH of the fermented mare's milk to 4.30 with 5M HCl, and treat at 37°C, 60rpm for 2h; then adjust the pH value to 7.0 with 5M NaOH, add 0.1% (w / v) trypsin, treat at 37°C, 60rpm for 2h, and then centrifuge at 4°C, 12000g for 15min, take the supernatant, and freeze-dry to obtain lyophilized powder;

[0033] 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 freeze-dried using a vacuum freeze dryer and further prepared into a 50 mg / mL solution with deionized water. The inhibition rate of each component on pancreatic lipase activity was determined. The results are shown in 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 separated solution of peak 1 had an inhibitory effect on pancreatic lipase activity. The HPLC chromatogram of this peak is shown in Figure 5 ,from Figure 5 It can be seen that the liquid phase results show a single peak, indicating that the separation is complete.

[0034] The chromatographic conditions of RP-HPLC were as follows: injection volume 1 mL, flow rate 2 mL / min, detection wavelength at 215 nm; mobile phase A contained 0.1% (v / v) trifluoroacetic acid (TFA) in deionized water, and mobile phase B contained 0.1% (v / v) TFA in acetonitrile (CH3 CN or C 2 H 3 N), gradient elution was performed using mobile phases A and B, and the elution program was: 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.

[0035] Example 2: LC-MS identification

[0036] The peak component No. 1 was identified by LC-MS, and the analysis time was 35 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: after each full scan (full scan), the fragment spectrum (MS2 scan) was collected; the mass spectrometry test raw file (Raw File) was searched with the corresponding database (Equuscaballus) using the software Mascot2.2, and finally the protein identification result was obtained;

[0037] 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 HAWF.

[0038] Experimental Example 3: Docking of small molecule peptide HAWF with pancreatic lipase

[0039] The crystal structure of pancreatic lipase (PDB number: 1ETH) was obtained from the RCSB Protein Data Bank database. HAWF was docked with pancreatic lipase using CB DOCK2, and the total binding energy between HAWF and pancreatic lipase was obtained to be -10.1 kcal / mol. The key amino acid residues and interaction forces between HAWF and pancreatic lipase were determined. Figure 7 As can be seen from the figure, the HAWF peptide can interact with 12 amino acid residues, and the interactions include hydrogen bonds, hydrophobic interactions and ionic interactions. The HAWF peptide can form hydrophobic interactions with substrate binding residues Phe216, Ieu154, ALa261, Arg57, Val260, Leu294, and Tyr115. It forms hydrogen bonds with catalytic residues Ser153 and Arg257, as well as salt bridges with the substrate His264 site and π-π stacking at the substrate Phe216 and Phe78 sites. In short, HAWF inhibits the activity of pancreatic lipase mainly by occupying the catalytic and substrate binding sites.

[0040] Example 4: Detection of the inhibitory activity of small molecule peptide HAWF on pancreatic lipase

[0041] 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 HAWF on pancreatic lipase was 94% at a concentration of 5 mg / mL.

Claims

1. A small molecule peptide derived from fermented mare's milk, with an amino acid sequence of His-Ala-Trp-Phe.

2. Use of the 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 have pancreatic lipase inhibitory activity.

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