Fermented mare milk-derived small molecule peptide and application thereof

Abstract

The application discloses a small-molecule peptide from fermented mare milk, which has an amino acid sequence of His-Ala-Trp-Phe and a molecular weight of 559.67 Da. The small-molecule peptide has pancreatic lipase inhibitory activity, can be applied to the preparation of a medicine for reducing high blood fat, and is simple to prepare, suitable for industrialized production and market promotion and application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a fermented horse milk-derived small-molecule peptide and application thereof. BACKGROUND

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

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

[0004] Another object of the application is to apply the fermented horse milk-derived small-molecule peptide to preparation of an anti-obesity or hyperlipidemia-preventing preparation.

[0005] The preparation comprises the small-molecule peptide as an active ingredient, and one or more pharmaceutically acceptable adjuvants can be added to improve the absorption effect of the drug or facilitate use, so that the preparation is in a suitable dosage form, such as a capsule or a pill, a powder, a tablet, a granule, an oral liquid, an injection, or the like, i.e., a pharmaceutically suitable dosage form. The preparation can be used to prepare a drug for treating or assisting in treating obesity or hyperlipidemia.

[0006] The object of the application is achieved by the following scheme.

[0007] 1. Fresh horse milk is sterilized at 65-115 DEG C for 2-15 min, inoculated with lactic acid bacteria and yeast, the inoculation amount is 0.1-10%, the fermentation temperature is 20-37 DEG C, the shaking speed is 50-300 rpm, and the fermentation time is 16-90 h; the lactic acid bacteria are Lactobacillus paracasei, and the yeast is Dekkaromyces hetero-thermophilus and Kazachstania unispora.

[0008] 2. After the fermentation of fresh horse milk is finished, 0.3% (w / v) pepsin is added, and the pH of the fermented horse milk is adjusted to 4.0-4.8 by 5M HCl, and then the mixture is treated at 30-38℃, 50-100rpm for 2-4h; then the pH is adjusted to 7.0 by 5M NaOH, and 0.1% (w / v) trypsin is added, and the mixture is treated at 37℃, 60rpm for 2-4h, and then the supernatant is obtained by centrifugation at 4℃, 12000g for 10-15min, and freeze-drying is carried out to obtain a freeze-dried powder;

[0009] 3. The freeze-dried powder is dissolved in double-distilled water to prepare a solution with a concentration of 0.05-5g / mL, the solution is filtered by using an ultrafiltration membrane with different molecular weight cut-off, different fractions of liquid are separated, and the pancreatic lipase inhibitory activity of each fraction is determined. The ultrafiltration component with the strongest pancreatic lipase inhibitory activity is selected for reverse phase high performance liquid chromatography (RP-HPLC) separation and purification. The peak with pancreatic lipase inhibitory activity after RP-HPLC purification is identified by liquid chromatography-mass spectrometry (LC-MS), the obtained peptide sequence is subjected to peptide activity prediction by BIOWARE database, molecular docking simulation and pancreatic lipase inhibitory activity prediction are carried out by using alpha-fold3 and CB-Dock2 databases, the peptide sequence with the best predicted activity is selected for solid-phase synthesis verification, and finally a novel molecular peptide with pancreatic lipase inhibitory activity is obtained.

[0010] The fermented horse milk-derived small molecule peptide HAWF of the present application can significantly inhibit the activity of pancreatic lipase, and can be applied to the preparation of drugs for treating or assisting in the treatment of hyperlipidemia, and the preparation is simple, suitable for industrialized production and market promotion and application. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 The results of pancreatic lipase inhibitory activity of the solution containing substances with different molecular weights after ultrafiltration cut-off;

[0012] Figure 2 The separation peak diagram of the component with a molecular weight of less than 3kDa after RP-HPLC separation;

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

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

[0015] Figure 5 The high performance liquid chromatogram of the separation liquid of peak No. 1;

[0016] Figure 6 The mass spectrometry identification diagram of the separation liquid of peak No. 1;

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

[0018] Figure 8 Detection results of small molecule peptide HAWF in inhibiting pancreatic lipase activity in vitro. DETAILED DESCRIPTION

[0019] The technical solutions of the present application are further described below by examples, but the content of the present application is not limited thereto. The methods in the present examples are all conventional methods unless otherwise specified. The materials, reagents and the like used are all obtained from commercial channels unless otherwise specified.

[0020] In the following examples, the pancreatic lipase solution is prepared with Tris-HCl buffer, the small molecule peptide solution is prepared with deionized water, and the p-NPB substrate solution is prepared with DMSO.

[0021] After mixing the pancreatic lipase solution and the small molecule peptide solution, preheat at 37°C for 10 min, then add the p-NPB substrate solution at 37°C and react for 15-30 min. Take out the reaction product, centrifuge at 5000g for 5 min, take the supernatant and detect the absorbance value at 405 nm (experimental group).

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

[0023] Control group: Tris-HCl buffer + PL + p-NPB, without small molecule peptide.

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

[0025] Add Orlistat as a positive control.

[0026] The reaction system is prepared according to Table 1. The experiment needs to be repeated three times, and the inhibition rate is calculated according to the following formula:

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

[0028]

[0029] × 100%.

[0030] Example 1: Obtaining of small molecule peptide

[0031] 1. Fresh mare milk was sterilized at 115°C for 10 min; after the third generation of activation, the heteroform of Dekkera, Kazachstania and Lactobacillus paracasei were inoculated into the sterilized fresh mare milk at a ratio of 2% for the yeast and 4% for the lactic acid bacteria, and then fermented at 37°C and 150 rpm for 48 h;

[0032] 2. 0.3% (w / v) pepsin was added to the fermented mare milk solution, the pH of the fermented mare milk was adjusted to 4.30 with 5M HCl, and the solution was treated at 37°C and 60 rpm for 2 h; then the pH was adjusted to 7.0 with 5M NaOH, 0.1% (w / v) trypsin was added, and the solution was treated at 37°C and 60 rpm for 2 h; then the solution was centrifuged at 4°C and 12000g for 15 min, the supernatant was taken, and freeze-dried to obtain a freeze-dried powder;

[0033] 3. The freeze-dried powder was dissolved in deionized water to prepare a 100 mg / mL solution, and the solution was filtered through ultrafiltration membranes with different cut-off values 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, and the inhibition rate of each component on pancreatic lipase activity was determined, as shown in Figure 1 From the figure, it can be seen that 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, and the results are shown in Figure 2 According to different time periods, the peaks in Figure 2 were divided into 7 parts, the separated solutions of different time periods were collected and freeze-dried after removing the mobile phase using a rotary evaporator, and each time period was configured into a 10 mg / mL solution with deionized water, and the inhibitory activity of different component solutions on pancreatic lipase was determined, as shown in Figure 3 From the figure, it can be seen that the component with the best inhibitory effect on pancreatic lipase activity is the separated solution of the 35-40 min time period, which was further separated by RP-HPLC, and the results are shown in Figure 4 The separated solutions of different peaks were collected and freeze-dried after removing the mobile phase using a rotary evaporator, and the inhibitory activity of different peak solutions on pancreatic lipase was determined, and the separated solution of peak No. 1 had an inhibitory effect on pancreatic lipase activity, and the high-performance liquid chromatogram of the peak is shown in Figure 5 From the Figure 5 , it can be seen that the liquid phase result shows a single peak, indicating that the separation is complete.

[0034] The chromatographic conditions for RP-HPLC were as follows: injection volume 1 mL, flow rate 2 mL / min, detection wavelength at 215 nm; mobile phase A was deionized water containing 0.1% (v / v) trifluoroacetic acid (TFA), and mobile phase B was acetonitrile (CH3CN or C2H3N) containing 0.1% (v / v) TFA. Gradient elution was performed using mobile phases A and B, with the following elution program: 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 fraction of peak 1 was identified by LC-MS, analysis time: 35 min. Detection mode: positive ion. The mass-charge ratio of small peptides and small peptide fragments was collected using the following method: fragment spectra were acquired after each full scan (MS2 scan); the raw mass spectrometry test file was retrieved from the corresponding database (Equuscaballus) using Mascot 2.2 software, and finally the protein identification results were obtained.

[0037] See Figure 6 The corresponding intensity and charge-to-mass ratio of the fragments can be obtained from the figure. Sequence alignment with the database confirms that the small molecule peptide is HAWF.

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

[0039] The crystal structure of pancreatic lipase (PDB ID: 1ETH) was obtained from the RCSB Protein Data Bank database. HAWF was docked with pancreatic lipase using CB DOCK2, yielding a total binding energy of -10.1 kcal / mol. The key amino acid residues and interaction forces between HAWF and pancreatic lipase were determined. (See [link to relevant documentation]). Figure 7 As shown in the figure, the HAWF peptide can interact with 12 amino acid residues, and these 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, and forms a salt bridge with the substrate His264 site and π-π stacking at substrate Phe216 and Phe78 sites. In summary, HAWF mainly inhibits pancreatic lipase activity by occupying catalytic and substrate-binding sites.

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

[0041] The small molecule peptide sequence was synthesized by Shanghai Biotech, with a purity of ≥98%. The obtained small molecule peptide was gradient dissolved to obtain 1, 2, 3, 4, and 5 mg / mL small molecule peptide solutions, and the inhibitory effect of different concentrations of small molecule peptide solutions on pancreatic lipase activity was detected. The results are shown in Figure 8 The inhibitory 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 milk, having an amino acid sequence of His-Ala-Trp-Phe.

2. Use of the small molecule peptide of claim 1 in the preparation of a medicament for preventing and treating obesity or hyperlipidemia.

Images