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 Met-Ser-Pro-Phe (MSPF), and the molecular weight of which is 480.571 Da. The small molecule peptide disclosed by the invention has the activity of inhibiting pancreatic lipase (PL), the inhibition rate on the pancreatic lipase is 61.44%, and the accumulation of the content of triglyceride (TG) in oleic acid-induced human liver cancer (HepG2) cells can be reduced, so that the small molecule peptide can be used for inhibiting the pancreatic lipase (PL). The small molecule peptide can be applied to preparation of preparations for treating or assisting in treating hyperlipidemia and obese people, is simple to prepare, and is suitable for industrial production and market popularization and application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a small molecule peptide derived from horse milk and its applications. Background Technology

[0002] With improved living conditions and changes in dietary structure, the global obese population is gradually increasing. Obesity has gradually become a serious global public health problem, causing not only physiological and metabolic abnormalities but also a series of psychological issues. Pancreatic lipase is a class of enzymes with lipolytic activity, capable of hydrolyzing 50-70% of triglycerides in daily diets, playing a crucial role in lipid metabolism. Therefore, the inhibition and regulation of pancreatic lipase may be an ideal target for the prevention and treatment of metabolic diseases such as hyperlipidemia and obesity. Orlistat, as a commercially available lipase inhibitor, has a good therapeutic effect on obesity, but it has side effects such as liver damage and adverse gastrointestinal symptoms. In recent years, naturally derived pancreatic lipase inhibitors have attracted much attention due to their structural diversity, low toxicity, and wide availability.

[0003] Exogenous bioactive peptides are protein hydrolysates with specific biological activities, typically prepared from biological proteins through enzymatic hydrolysis, bioengineering, and other techniques. They are a general term for small molecule peptides composed of 20 natural amino acids in different compositions and arrangements. Numerous studies have demonstrated that small molecule peptides in fermented mare's milk can regulate bodily functions and possess various biological activities such as antioxidant, immune-enhancing, antibacterial, and blood pressure-lowering effects, while also exhibiting certain nutritional and medicinal value. Based on their safety and efficacy, bioactive peptides have emerged as an ideal biological resource with the potential to be used in food, health products, and pharmaceuticals. Summary of the Invention

[0004] This invention provides a small molecule peptide derived from fermented mare's milk, with the amino acid sequence Met-Ser-Pro-Phe (MSPF) and a molecular weight of 480.571 Da. It has pancreatic lipase inhibitory activity and can be used to prepare pancreatic lipase inhibitors.

[0005] Another objective of this invention is to apply the above-mentioned fermented mare milk-derived small molecule peptides in the preparation of anti-obesity or anti-hyperlipidemia preparations.

[0006] The components (or active ingredients) of the formulations described in this invention are the aforementioned small molecule peptides. One or more pharmaceutical or food-acceptable excipients may also be added to improve the absorption of the drugs or foods or to facilitate use, thereby creating suitable dosage forms, such as capsules, pills, powders, tablets, granules, oral liquids, and injections. In other words, they can be formulated into pharmaceutically suitable dosage forms or suitable methods of consumption in the food industry. They can be applied to the preparation of functional foods, health products, and drugs for the treatment or adjuvant treatment of obesity or the prevention and treatment of hyperlipidemia.

[0007] The objective of this invention is achieved through the following solution: 1. Fresh mare's milk is sterilized at 85-105℃ for 5-15 minutes. After the temperature drops to room temperature, lactic acid bacteria and yeast are inoculated at an inoculation rate of 0.2-10%. The fermentation temperature is 25-40℃, the shaking speed is 100-300 rpm, and the fermentation time is 12-48 hours. The lactic acid bacteria are Lactobacillus helveticus, and the yeast is Kluyveromyces martensii. 2. After the fermentation of fresh mare's milk is completed, an in vitro gastrointestinal simulation is performed. 0.3% (w / v) pepsin is added to the fermented mare's milk, and then the pH is adjusted to 4.0–4.8 with 0.1M HCl. The mixture is then treated at 30–38℃ and 50–100 rpm for 2–4 hours. Next, the pH is adjusted to 7.0 with 0.1M NaOH, and 0.1% (w / v) trypsin is added. The mixture is then treated at 37℃ and 60 rpm for 2–4 hours. After treatment, the enzyme is inactivated, and the supernatant is collected by centrifugation at 4℃ and 12000g for 10–15 minutes. 3. The supernatant was centrifuged using ultrafiltration tubes with cutoffs of 10 kDa and 3 kDa to obtain solutions containing components with molecular weights >10 kDa, 3-10 kDa, and <3 kDa. The solutions containing the three components were freeze-dried separately, and the freeze-dried powders were collected. The freeze-dried powders were then dissolved in distilled water, and the pancreatic lipase inhibitory activity of each component was detected. The component with the highest inhibition rate was selected for separation and purification by reversed-phase high-performance liquid chromatography (RP-HPLC). The peak with pancreatic lipase inhibitory activity after RP-HPLC purification was selected for LC-MS identification. The peptide sequence obtained after identification was used for activity prediction in BIOPEP. Molecular docking simulation and pancreatic lipase inhibitory activity prediction were performed using the α-fold3 and CB-Dock2 databases. The component with the best predicted activity was verified by solid-phase synthesis, and finally, the small molecule peptide of the present invention was obtained.

[0008] The fermented mare milk-derived small molecule peptide Met-Ser-Pro-Phe of this invention can inhibit pancreatic lipase activity, improve the accumulation of triglycerides in oleic acid-induced HepG2 cells, and reduce the accumulation of lipid droplets in HepG2 cells induced by oleic acid. The small molecule peptide of this invention can be used to prepare drugs or foods that alleviate or assist in the treatment of obesity or hyperlipidemia. Attached Figure Description

[0009] Figure 1 The results show the inhibitory activity of solutions containing substances of different molecular weights after ultrafiltration on pancreatic lipase. Figure 2 A schematic diagram of the separation peaks after RP-HPLC separation of components with molecular weight <3kDa; Figure 3The results show the pancreatic lipase inhibitory activity of the separation solution corresponding to the separation peak after RP-HPLC separation. Figure 4 The RP-HPLC separation results are for the separated liquid over a period of 35-40 minutes. Figure 5 The high-performance liquid chromatogram of the separated solution for peak M1; Figure 6 The mass spectrometry chromatogram of the separated solution for peak M1; Figure 7 This is a schematic diagram of the molecular docking between the small molecule peptide MSPF and pancreatic lipase. Figure 8 The results of in vitro assays for the inhibition of pancreatic lipase activity by the small molecule peptide MSPF; Figure 9 Results of the effect of small molecule peptide MSPF on the activity of HepG2 cells; Figure 10 The effect of small molecule peptide MSPF on oleic acid-induced triglyceride accumulation in HepG2 cells. Detailed Implementation

[0010] The present invention will be further illustrated by the following examples, but the protection of the present invention is not limited to the content described. Unless otherwise specified, the methods in this embodiment are operated according to conventional methods, and the experimental equipment, reagents and other materials used are obtained from commercial sources unless otherwise specified.

[0011] The method for detecting pancreatic lipase inhibitory activity in the examples is as follows: Dissolve pancreatic lipase in 0.1M Tris-HCl (pH 8.0) buffer to prepare a 2 mg / mL solution, and add 10 µL of p-NPB to 5 mL of DMSO to prepare a solution. After mixing pancreatic lipase (PL) solution and small molecule peptide solution, the mixture was preheated at 37°C for 10 min. After preheating, p-NPB substrate solution was added at 37°C and reacted for 15-30 min. The reaction product was then centrifuged at 5000g for 5 min, and the supernatant was collected and its absorbance value was measured at 405 nm (experimental group). A blank control group was also set up: Tris-HCl buffer + small molecule peptide + p-NPB; Control group: Tris-HCl buffer + PL + p-NPB, without small molecule peptides; Control group: Tris-HCl buffer + p-NPB, without small peptides and PL; Positive control blank group: Tris-HCl buffer + orlistat + p-NPB; Positive control group: PL + Orlistat + p-NPB The reaction system was prepared according to Table 1. The experiment was repeated 3 times, and the pancreatic lipase inhibition rate was calculated according to the following formula. Table 1. Reaction system for pancreatic lipase activity assay

[0012] ; Experimental Example 1: Obtaining Small Molecule Peptides (1) Fresh mare milk was sterilized at 105℃ for 10 min; activated third-generation Lactobacillus helveticus and Kluyveromyces roxburghii were inoculated into the sterilized mare milk after cooling to room temperature, with yeast at 2% by volume and lactic acid bacteria at 4% by volume. Then fermented at 37℃ and 150 rpm for 48 h. (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 0.1M HCl, and treat it at 37℃ and 60 rpm for 2 h; then adjust the pH of the fermented mare's milk to 7.0 with 0.1M NaOH, add 0.1% (w / v) trypsin, treat it at 37℃ and 60 rpm for 2 h, then inactivate the enzyme by water bath at 90℃ for 10 min, and finally centrifuge at 4℃ and 12000g for 10 min and take the supernatant. (3) The supernatant was centrifuged using ultrafiltration tubes with different cut-off values ​​of 10 kDa and 3 kDa to obtain three different components: >10 kDa, 3-10 kDa, and <3 kDa. These three components were freeze-dried using a vacuum freeze dryer and further dissolved in deionized water to prepare a solution with a concentration of 25 mg / mL. The inhibition rate of each component on pancreatic lipase activity was determined, and the results are shown in […]. Figure 1 As shown in the figure, the fraction with the highest pancreatic lipase inhibitory activity is the one with a molecular weight <3kDa. This fraction was further separated using RP-HPLC, and the results are as follows. Figure 2 As shown, according to different time periods Figure 2 The peak was divided into five parts. Separates from different time periods were collected, and the mobile phase was removed using a rotary evaporator. The recovered liquid was then freeze-dried, and the freeze-dried components from each time period were prepared into 10 mg / mL solutions using deionized water. The inhibitory activity of different component solutions on pancreatic lipase was measured. The results are shown in [Figure Number]. Figure 3 As shown in the figure, the fraction with the highest pancreatic lipase inhibitory activity was the fraction obtained in the 35-40 min time period. This fraction was further separated by RP-HPLC, and the results are shown in the figure. Figure 4The separated solutions of different peaks were collected again, and after removing the mobile phase using a rotary evaporator, they were freeze-dried. The inhibitory activity of the solutions with different peaks on pancreatic lipase was measured. The results showed that the solution with peak M1 had the highest inhibitory activity on pancreatic lipase. The high-performance liquid chromatogram of this peak is shown in [Figure number missing]. Figure 5 ,from Figure 5 The liquid phase result shows a single peak, indicating that the separation is complete.

[0013] The chromatographic conditions for RP-HPLC were as follows: injection volume 1000 μL, 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.

[0014] Example 2: LC-MS Identification The M1 peak component was identified by LC-MS, analysis time: 30 min. Detection mode: positive ion. The mass-charge ratio of small peptides and small peptide fragments was acquired using the following method: 10 fragment spectra were acquired for each full scan (MS2 scan); the raw mass spectrometry file was retrieved from the relevant database (Equus caballus) using Mascot 2.2 software, and finally, the protein identification results were obtained.

[0015] See results Figure 6 The corresponding intensity and mass-charge ratio of the fragments can be obtained from the figure. Sequence alignment with the database confirms that the small molecule peptide is MSPF.

[0016] Experimental Example 3: Molecular docking of small molecule peptide MSPF with pancreatic lipase A three-dimensional model of pancreatic lipase (PL) was downloaded from the RCBSPDB protein database. The 3D structure of the small peptide was plotted using α-fold3, and an NM2 force field was added to minimize its energy. Flexible molecular docking was performed using Gold 5.3.0. Ligands were extracted from the acceptor, water molecules were removed, and polar hydrogen was added. The docking model was screened using the Gold score system. The results showed that the total binding energy of MSPF with pancreatic lipase was -8.0 kcal / mol, and the key amino acid residues and interaction forces for its interaction with pancreatic lipase were identified. Visual analysis of the docked molecular complex was performed using Moe 2019.10 software. The results are shown in [Figure 1]. Figure 7As shown in the figure, MSPF exhibits both hydrophobic and ionic interactions. MSPF forms 11 hydrophobic interactions with the substrate (P181, I210, F216, Y115, F18, 179, V260, R257, W253, L265, L154) and 3 ionic interactions (H152, H264, R257), forming a weak hydrogen bond (H152) with the catalytic residue. In summary, MSPF primarily inhibits pancreatic lipase activity by occupying substrate binding sites and catalytic sites.

[0017] Example 4: Detection of the inhibitory activity of small molecule peptide MSPF on pancreatic lipase The small peptide sequence was synthesized by Shanghai Sangon Biotech with a purity ≥98%. The obtained small peptides were then subjected to gradient dissolution to obtain small peptide solutions of 100, 200, 300, 400, and 500 μg / mL. The inhibitory effects of different concentrations of small peptide solutions on pancreatic lipase activity were detected. Results are shown below. Figure 8 The small molecule peptide MSPF inhibited pancreatic lipase by 61.44% at a concentration of 400 μg / mL.

[0018] Experimental Example 5: Functional Evaluation of Small Molecule Peptide MSPF (1) Detection of the effect of MSPF small molecule peptides on cell viability by CCK-8 assay HepG2 cells cultured to 80% plating were digested with trypsin and then suspended in DMEM medium containing 10% FBS. Cells were then cultured at a concentration of 1.0 × 10⁶ cells / mL. 5 Add the reagent to each well of a 96-well plate, then add DMEM medium containing small molecule peptides at concentrations of (0 mg / L, 5 mg / L, 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L) to culture HepG2 cells. Culture in a cell culture incubator at 37℃ and 5% CO2 for 24 h. Then add 10 μL CCK-8 reagent to each well of each group and culture in a cell culture incubator for 40 min. After culture, measure the absorbance at 450 nm. Perform three replicates for each group. Results are shown below. Figure 9 ,Depend on Figure 9 It can be seen that the small molecule peptide MSPF has almost no toxic side effects on HepG2 cells; among them, the small molecule peptide with a concentration of 5 mg / L has the least impact on cell activity, so this concentration was chosen for subsequent experiments. (2) Effect of small molecule peptide MSPF on triglyceride accumulation in HepG2 cells HepG2 cells cultured to 80% plating were digested with trypsin and then suspended in DMEM medium containing 10% FBS. Cells were then cultured at a concentration of 1.0 × 10⁶ cells / mL. 5 Add it to the 96-well plate and set the following groups: Model group (DC): HepG2 cells were cultured in DMEM medium containing 0.5 mmol / L oleic acid; Normal group (NC): HepG2 cells were cultured in DMEM medium; Positive control group (PC): HepG2 cells were cultured in DMEM medium containing 25 μg / mL orlistat and 0.5 mmol / L oleic acid; Small molecule peptide group (MSPF): HepG2 cells were cultured in DMEM medium containing 5 mg / L small molecule peptides and 0.5 mmol / L oleic acid; After culturing the cells in each group at 37℃ and 5% CO2 for 24 h, the original culture medium was discarded. The cells were washed twice with PBS, digested with trypsin, and centrifuged at 3000 rpm for 5 min at 4℃. 200 μL of cell lysis buffer was added to each group, and the cells were lysed on ice for 30 min. After lysis, the cells were centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was collected, and the triglyceride content in the supernatant was determined using a triglyceride assay kit from Nanjing Jiancheng Biotechnology Co., Ltd. (referring to the kit instructions for water samples). The protein concentration of the precipitate was determined using a Beyotime BCA protein assay kit. Each group was performed in triplicate. Results are shown below. Figure 10 As shown in the figure, the small molecule peptide MSPF can significantly improve the accumulation of triglycerides in oleic acid-induced hyperlipidemic HepG2 cells.

Claims

1. A small molecule peptide derived from horse milk, the amino acid sequence of which is Met-Ser-Pro-Phe.

2. The use of the horse milk-derived small molecule peptide according to claim 1 in the preparation of anti-obesity or anti-hyperlipidemia preparations.

3. The application according to claim 2, characterized in that: Horse milk-derived small molecule peptides have pancreatic lipase inhibitory activity.

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