Milk-derived small molecule peptide and application thereof

Abstract

The invention discloses a milk-derived small molecule peptide, the amino acid sequence of the milk-derived small molecule peptide is Lys-Pro-Pro-Phe-Pro-Lys (KPPFPK), and the molecular weight of the milk-derived small molecule peptide is 712.86 Da. The milk-derived small molecule peptide disclosed by the invention has pancreatic lipase inhibitory activity, and the pancreatic lipase inhibition rate is 62.43%; the accumulation of the content of triglyceride (TG) in oleic acid induced human hepatoma cells (Hepg2) can be reduced; the small molecule peptide can be applied to preparation of preparations for treating or assisting in treating obesity or preventing and treating hyperlipidemia, and is simple to prepare and suitable 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 milk-derived small molecule peptide and application thereof in preparing an anti-obesity or hyperlipidemia prevention and treatment preparation. Background Art

[0002] Obesity not only causes mobility problems for the patient, but also causes many diseases such as chronic inflammation, cardiovascular disease and type 2 diabetes, and even worse, may lead to death. In short, obesity seriously affects the patient's physical health and quality of life.

[0003] The occurrence of overweight and obesity is usually described as abnormal or excessive accumulation of triglycerides (TG) in fat cells due to an imbalance between energy intake and energy expenditure. An unhealthy lifestyle may lead to a variety of health conditions, including cardiovascular disease, diabetes, musculoskeletal diseases, and various types of cancer. Therefore, people are paying more and more attention to obesity and ways to treat or prevent obesity. Currently, the FDA-approved drugs for the treatment of obesity mainly include diethylpropion, phentermine, orlistat, etc., but drug treatment is always accompanied by certain side effects, such as thirst, constipation, dizziness, insomnia, mild gastrointestinal dysfunction, and obstruction of the absorption of some fat-soluble vitamins. In comparison, food-derived pancreatic lipase inhibitory peptides have the advantages of simple preparation, low cost, and few side effects. As people tend to prefer preventive health care and diet therapy, this application has broad market prospects. Summary of the invention

[0004] The invention provides a small molecule peptide derived from bovine whey fermentation. The amino acid sequence of the small molecule peptide is Lys-Pro-Pro-Phe-Pro-Lys (KPPFPK) and the molecular weight is 712.86 Da. The small molecule peptide is used to prepare a pancreatic lipase inhibitor, and the results show that the small molecule peptide has pancreatic lipase inhibitory activity.

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

[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) Activation of bacterial strains: Lactobacillus paracasei and yeast were inoculated into MRS and YPD culture media, respectively, with an inoculation amount of 1-10%, a fermentation temperature of 20-37° C., a shaking speed of 100-300 rpm, a culture time of 8-24 h, and activation for 2-3 generations; the lactic acid bacteria were Lactobacillus paracasei, and the yeast was Dekkera heterozygosaccharomyces;

[0009] (2) Preparation of fermented whey: sterilize the whey solution at 55-105°C for 1-30 min, and when the temperature drops to room temperature, inoculate activated lactic acid bacteria and yeast; the inoculation amount is 1-10%, the fermentation temperature is 20-37°C, the shaking speed is 100-300 rpm, and the fermentation time is 12-50 h. After the fermentation is completed, add pepsin to the fermented whey solution, adjust the pH of the whey solution after the fermentation to 4-4.7 with 5 mol / L hydrochloric acid, and shake at 60 rpm and 37°C for 2 h; add 5 mol / L sodium hydroxide to adjust the pH to 7.0, add trypsin, shake at 60 rpm and 37°C for in vitro simulation of enzymatic hydrolysis for 2 h, then centrifuge to obtain the supernatant, and use freeze-drying technology to obtain freeze-dried powder;

[0010] (3) Take an appropriate amount of lyophilized powder and dissolve it in deionized water to prepare a solution of 5 to 200 mg / mL. Use three ultrafiltration cutoff tubes with ultrafiltration cutoff membranes of different molecular weights to filter the prepared solution to obtain liquids of different components. Determine the pancreatic lipase inhibitory activity of the liquids of different components. The ultrafiltration component with the best inhibitory activity is separated and purified by reverse HPLC (RP-HPLC). The peak separated and purified by RP-HPLC is subjected to the pancreatic lipase inhibitory activity determination. Then, the peak with the best inhibitory effect on pancreatic lipase activity is subjected to LC-MS screening of peptides. The identified peptide sequence is used to predict whether the peptide has biological activity using PetideRanker in BIOWARE. Subsequently, α-flod3 is used to predict the pancreatic lipase inhibitory activity. The peptide sequence with the best predicted activity is selected for synthesis verification to obtain the small molecule peptide of the present invention.

[0011] The fermented whey small molecule peptide sequence disclosed in the present invention is KPPFPK, which can significantly inhibit pancreatic lipase activity and improve the accumulation of triglyceride content in Hepg2 cells induced by oleic acid; the small molecule peptide of the present invention can be used to prepare medicines or foods for alleviating or assisting in the treatment of epidemic diseases such as obesity and hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results are the results of the inhibitory activity of the solutions containing substances of different molecular weights on pancreatic lipase (PL) after ultrafiltration retentate;

[0013] Figure 2It is a schematic diagram of peak separation after RP-HPLC separation of a component solution with a molecular weight less than 3 kDa;

[0014] Figure 3 The results of the inhibitory activity test on pancreatic lipase of the five peaks after RP-HPLC separation;

[0015] Figure 4 Schematic diagram of the separation peaks after RP-HPLC separation of the separation liquid in the 25-30min time period;

[0016] Figure 5 is the HPLC chromatogram of the No. 2 peak separation solution;

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

[0018] Figure 7 Schematic diagram of molecular docking between small molecule peptide KPPFPK and pancreatic lipase;

[0019] Figure 8 Schematic diagram of the inhibitory activity of small molecule peptide KPPFPK on pancreatic lipase at different concentrations;

[0020] Fig. 9 The results show the effects of different concentrations of small molecule peptide KPPFPK on the activity of Hepg2 cells;

[0021] Fig.10 These are the results of the effect of small molecule peptide KPPFPK on oleic acid-induced triglyceride accumulation in Hepg2 cells. 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 p-NPB substrate solution was added to react for 5 to 30 min, and then taken out 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 + p-NPB, 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] Example 1: Acquisition of small molecule peptides

[0034] (1) Preparation of fermented whey solution: whey powder was dissolved in deionized water to prepare a whey solution with a mass volume concentration of 7%, and sterilized at 65° C. for 5 min. After the temperature dropped to room temperature, three generations of activated Dekkera atypical and Lactobacillus paracasei were inoculated into the whey solution at an inoculation ratio of 2% and 4%, respectively, and then fermented at 30° C. and 150 rpm for 48 h;

[0035] (2) Pretreatment of fermented whey solution: add 0.3% (W / V) pepsin to the fermented whey solution, adjust the pH of the whey solution after fermentation to 4.30 with 5 mol / L hydrochloric acid, and shake at 60 rpm and 37°C for 2 h; add 5 mol / L sodium hydroxide to adjust the pH to 7.0, add 0.1% (W / V) trypsin and shake at 60 rpm and 37°C for 2 h to simulate enzymatic hydrolysis in vitro, then centrifuge at 4°C and 12000g for 15 min to obtain the supernatant, and obtain freeze-dried powder using freeze-drying technology;

[0036] (3) dissolving the lyophilized powder of step (2) in deionized water to prepare a 100 mg / mL solution, filtering the component using two ultrafiltration membranes with different retention amounts of 10 kDa and 3 kDa to obtain three different components of >10 kDa, 3-10 kDa, and <3 kDa; freeze-drying the three components using a vacuum freeze dryer and further preparing them into a 50 mg / mL solution using deionized water, and determining the inhibition rate of each component on pancreatic lipase activity;

[0037] Results Figure 1 ,from Figure 1 It can be seen that the component solution with the best inhibitory effect on pancreatic lipase activity is the component solution with a molecular weight of <3 kDa. The component solution was further separated using P-HPLC, and the results are as follows Figure 2As shown, according to different time periods Figure 2 The peaks in the sample were divided into five 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 ,from Figure 3 It can be seen that the component with the best inhibitory effect on pancreatic lipase activity is the separated liquid in the 25-30min 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 2 had the best 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 purification is complete.

[0038] 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.

[0039] Experimental Example 2: LC-MS Identification

[0040] 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.

[0041] 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 sequence is KPPFPK.

[0042] Experimental Example 3: Molecular docking of small molecule peptide KPPFPK and pancreatic lipase

[0043] The corresponding pancreatic lipase 3D model was downloaded from the RCBSPDB protein database. Alphafold 3 was used to draw the 3D structure of the small molecule peptide, and the NM2 force field was added to minimize its energy. CB-DOCK2 was used for flexible molecular docking, ligand extraction of the receptor was performed, water molecules were removed, polar hydrogen was added, and docking model screening was performed using its scoring system. The docking molecular complex was visualized and analyzed. The results are shown in Figure 7 ,As can be seen from the figure, the hydrophobic interaction binding sites of KPPFPK and pancreatic lipase are D390, Q29, A40, R38, and I249. The N-terminal domain (residues 1-335) with an α / β-hydrolase fold carries the active site, while the C-terminal domain (residues 336-449) forms a β-barrel structure and provides the main lipase binding site.

[0044] Experimental Example 4: Determination of the inhibitory activity of small molecule peptide KPPFPK on pancreatic lipase

[0045] The small molecule peptide sequence was synthesized by Shanghai Shenggong Biological Co., Ltd. with a purity of ≥98%. The obtained small molecule peptide was gradiently dissolved to obtain 1, 2, 3, 4, and 5 mg / mL small molecule peptide solutions. The inhibition of pancreatic lipase activity by small molecule peptide solutions of different concentrations was tested. The results are shown in Figure 8 The inhibition rate of small molecule peptide KPPFPK on pancreatic lipase was 62.43% at a concentration of 2 mg / mL.

[0046] Experimental Example 5: Functional evaluation of KPPFPK small molecule peptide

[0047] (1) Establishment of a high-lipid model of HepG2 cells induced by oleic acid (OA)

[0048] 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;

[0049] The model group (DC) cultured HepG2 cells with DMEM medium containing 0.5 mmol / L oleic acid;

[0050] The negative control group (NC) cultured HepG2 cells with DMEM medium without 0.5 mmol / L oleic acid;

[0051] 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;

[0052] The positive control group (PC) used DMEM medium containing 0.5mmol / L oleic acid and 25μmol / L orlistat to culture HepG2 cells;

[0053] For all groups, subsequent experiments were performed on cells after 24 h of culture;

[0054] (2) MTT assay to detect the effect of KPPFPK small molecule peptide on cell activity

[0055] 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 Add to 96-well plate, then add small molecule peptide solution with concentration of 1-5mg / mL, and set up control without adding small molecule peptide, culture in cell culture incubator at 37℃, 5% CO2 for 24h, then add 10Μ MTT reagent to each well of each group, culture in cell culture incubator for 4h, detect absorbance value at OD 490, do 3 biological parallels for each group, the results are shown in Fig. 9 ,As can be seen from the figure, the small molecule peptide KPPFPK has no toxic side effects on HepG2 cells;

[0056] (3) Effect of small molecule peptide KPPFPK on triglyceride accumulation in HepG2 cells

[0057] 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 the experimental group in 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 biological parallels were performed for each group. The results are shown in the table. Fig.10 As can be seen from the figure, the small molecule peptide KPPFPK can significantly improve the accumulation of triglyceride content in oleic acid-induced high-fat HepG2 cells.

Claims

1. A milk-derived small molecule peptide, whose amino acid sequence is Lys-Pro-Pro-Phe-Pro-Lys.

2. Use of the 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: Milk-derived small molecule peptides have pancreatic lipase inhibitory activity.

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