Cow milk clean-source small molecule peptide and application thereof

Abstract

The invention discloses a cow milk clear source small molecule peptide, the amino acid sequence of which is Trp-Thr-Pro-Tyr-Arg (WTPYR), and the molecular weight of which is 721.80 Da. The small molecule peptide disclosed by the invention has angiotensin converting enzyme (ACE) inhibitory activity, can be applied to preparation of preparations for preventing and treating hypertension, provides effective active ingredients for developing foods and products with antihypertensive efficacy and without side effects, and has a good application prospect. And the small molecule peptide has the characteristics of simple preparation and safety, and is suitable for industrial production and market popularization and application.

Description

Technical Field

[0001] This invention is designed in the field of biotechnology, specifically a bovine whey-derived small molecule peptide and its application in the preparation of drugs for the prevention and / or treatment of hypertension. Background Technology

[0002] Hypertension is one of the major risk factors for cardiovascular disease. While the exact cause of hypertension is unclear, discussions focus on its risk factors, including genetics, age, and unhealthy lifestyle habits. 70-80% of hypertension cases are related to unhealthy lifestyles. The risk of developing hypertension increases with the accumulation of these risk factors. Angiotensin-converting enzyme (ACE) plays a crucial role in its pathogenesis.

[0003] Angiotensin-converting enzyme (ACE) increases blood pressure by converting angiotensin I to angiotensin II, inactivating bradykinin (which has a vasodilatory effect), and promoting aldosterone secretion. Therefore, inhibiting ACE activity can treat hypertension. Commonly used ACE inhibitors include enalapril, captopril, benazepril, lamivudine, and lixinpril, which have significant therapeutic effects on hypertension. However, long-term use can easily lead to kidney damage, hyperkalemia, allergic reactions, and fetal maldevelopment.

[0004] Compared with chemically synthesized angiotensin-converting enzyme (ACE) inhibitors, dietary peptide-based ACE inhibitors offer advantages such as higher safety, easier absorption, and fewer side effects, providing a new approach for the prevention and treatment of hypertension. Currently, many bioactive collagen peptide products generally have large molecular weights, resulting in low levels of effective active ingredients and low product purity. Summary of the Invention

[0005] This invention provides a small molecule peptide produced after fermentation of bovine whey. The amino acid sequence of this small molecule peptide is Trp-Thr-Pro-Tyr-Arg(WTPYR), and the molecular weight is 721.80 Da. It has angiotensin-converting enzyme (ACE) inhibitory activity and can be used to prepare angiotensin-converting enzyme (ACE) inhibitors.

[0006] Another objective of this invention is to apply bovine whey-derived small molecule peptides in the prevention and / or treatment of hypertension.

[0007] The components (or active ingredients) of the formulation of 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 drug or food or to facilitate its use, and to formulate a suitable dosage form, such as capsules or pills, powders, tablets, granules, oral liquids, and injections, etc., that is, to formulate a pharmaceutically suitable dosage form or a food-appropriate consumption method; it can be applied to the preparation of functional foods, health products, and drugs for the treatment or adjuvant treatment of hypertension.

[0008] The objective of this invention is achieved through the following solution: 1. Inoculate yeast into YPD liquid medium and culture at 20-40 ℃ and 60-250 rpm for 8-46 h, subculturing 2-3 times for later use; inoculate lactic acid bacteria into MRS liquid medium and culture at 25-40 ℃ and 0-150 rpm for 20-72 h, subculturing 2-3 times for later use; the lactic acid bacteria mentioned above are Lactobacillus helveticus, and the yeast is Kluyveromyces martensii; 2. Sterilize the bovine whey solution at 90–105 °C for 5–10 min. When the temperature drops to room temperature, inoculate with 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. Then, add pepsin to the fermented whey solution and adjust the pH of the whey solution after fermentation to 4–4.7 with 0.1 mol / L hydrochloric acid. Shake at 60 rpm and 37 °C for 2 h. Then, add 0.1 mol / L sodium hydroxide to adjust the pH to 7.0. Add trypsin and simulate enzymatic hydrolysis in vitro at 60 rpm and 37 °C for 2 h. Then, centrifuge and collect the supernatant. Freeze-dry the supernatant to obtain the lyophilized fermented whey powder. 3. Dissolve the lyophilized fermented whey powder in deionized water to prepare a solution with a concentration of 5–200 mg / mL. Filter the prepared solution using three ultrafiltration membranes with different molecular weight cut-offs to obtain liquids with different components. Measure the angiotensin-converting enzyme (ACE) inhibitory activity of each component liquid. Separate and purify the ultrafiltration component with the best inhibitory activity using reverse high-performance liquid chromatography (RP-HPLC). Measure the ACE inhibitory activity of the purified peak obtained by RP-HPLC. Then, screen peptides by LC-MS for the peak with the best inhibitory effect on ACE activity. Predict the biological activity of the identified peptide sequences using PetideRanker in BIOWARE. Subsequently, predict the ACE inhibitory activity using α-flod3. Select the peptide sequence with the best predicted activity for synthesis verification to obtain the small molecule peptide of this invention.

[0009] The small molecule peptide Trp-Thr-Pro-Tyr-Arg (WTPYR) derived from bovine whey protein in this invention can significantly inhibit angiotensin-converting enzyme (ACE) activity. This small molecule peptide has the potential to be used in the preparation of drugs or foods for the prevention and / or treatment of hypertension. Moreover, the preparation of the small molecule peptide is simple, safe and without side effects, and it is suitable for industrial production and market promotion. Attached Figure Description

[0010] Figure 1 The results show the inhibitory activity of solutions containing substances of different molecular weights after ultrafiltration on angiotensin-converting enzyme (ACE). Figure 2 This is a schematic diagram of peak separation after RP-HPLC for a component solution with a molecular weight <3 kDa. Figure 3 The results of RP-HPLC separation of five peaks for the inhibition of angiotensin-converting enzyme (ACE) in a solution of a component with a molecular weight <3 kDa are presented. Figure 4 This is a schematic diagram of the separation peaks after RP-HPLC separation of the separated liquid over a time period of 10-15 min. Figure 5 The results show the inhibitory activity of the separated solution against angiotensin-converting enzyme (ACE) over a 10-15 min time period after RP-HPLC separation. Figure 6 The high-performance liquid chromatogram of the separated solution from peak A2; Figure 7 The mass spectrometry chromatogram of the separated solution for peak A2; Figure 8 This is a schematic diagram of the molecular docking between the small molecule peptide WTPYR and angiotensin-converting enzyme (ACE). Figure 9 This is a schematic diagram illustrating the inhibitory activity of the small molecule peptide WTPYR at different concentrations on angiotensin-converting enzyme (ACE). Specific implementation methods

[0011] 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.

[0012] The angiotensin-converting enzyme (ACE) inhibition rate was detected in the following examples: 1 mM N-[3-(2-furanyl)acryloyl]-L-phenylalanylglycylglycine (FAPPG) was used as the substrate; 0.1 U / mL angiotensin-converting enzyme (ACE) was used; HEPES was used as the buffer matrix (1.901 g HEPES reagent and 1.755 g NaCl reagent were weighed, dissolved in an appropriate amount of deionized water, and then the pH of the buffer was adjusted to 8.3 with NaOH solution, and the volume was brought to 100 mL for later use); Different concentrations of the small molecule peptide WTPYR sample (100, 200, 300, 400, 500 μg / mL) were added sequentially to 96-well plates. Reagents were added according to the reaction system in Table 1. A blank control group was also included. The absorbance of the reaction system at 340 nm was measured using a microplate reader and recorded. After incubating the reaction system in a shaker at 37 ℃ for 30 min, the absorbance of the reaction system at 340 nm was measured again using a microplate reader. The experiment was repeated three times. The ACE inhibition rate of the sample was calculated using the following formula. ; Table 1 ; Example 1: Obtaining Small Molecule Peptides 1. Dissolve bovine whey powder in deionized water to prepare a whey solution with a mass-volume concentration of 7%. Sterilize at 95 ℃ for 5 min. After the temperature drops to room temperature, inoculate the activated third-generation Kluyveromyces martensii and Lactobacillus helveticus into the whey solution at an inoculation ratio of 2% and 4% by volume, respectively. Then ferment at 30 ℃ and 150 rpm for 48 h. 2. Add 0.3% (w / v) pepsin to the whey solution after fermentation, adjust the pH of the whey solution to 4.30 with 0.1 mol / L hydrochloric acid, and shake in a shaker at 60 rpm and 37 ℃ for 2 h. Then add 0.1 mol / L sodium hydroxide to adjust the pH to 7.0, add 0.1% (w / v) trypsin, and simulate enzymatic hydrolysis in vitro in a shaker at 60 rpm and 37 ℃ for 2 h. Then centrifuge at 4 ℃ and 12000 g for 15 min and collect the supernatant. Freeze-dry the supernatant to obtain the fermented whey freeze-dried powder. 3. Dissolve the lyophilized whey powder from step 2 in deionized water to obtain a solution with a concentration of 100 mg / mL. Filter this solution using two ultrafiltration membranes with different rejection ratios of 10 kDa and 3 kDa to obtain three different components: >10 kDa, 3-10 kDa, and <3 kDa. Freeze-dry the three components using a vacuum freeze dryer and further prepare a solution with a concentration of 50 mg / mL using deionized water. Determine the inhibition rate of each component on angiotensin-converting enzyme (ACE) activity. Results are shown below. Figure 1 ,from Figure 1 It was found that the solution containing a molecular weight <3 kDa showed the best inhibitory effect on angiotensin-converting enzyme (ACE) activity. This solution 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 fractions. Separates from different time points were collected, and after removing the mobile phase using a rotary evaporator, they were freeze-dried. The freeze-dried fractions from each time point were then prepared into 10 mg / mL solutions using deionized water. The inhibitory activity of different fraction solutions on angiotensin-converting enzyme (ACE) was measured. The results are shown in [Figure number missing]. Figure 3 ,from Figure 3 It was found that the fraction with the best inhibitory effect on angiotensin-converting enzyme (ACE) activity was the separation solution within the 10-15 min time period. This fraction was further separated using RP-HPLC, and the results are shown below. Figure 4 After collecting the separated solutions with different peaks and removing the mobile phase using a rotary evaporator, the solutions were freeze-dried. The inhibitory activity of the solutions with different peaks on angiotensin-converting enzyme (ACE) was measured. The results are shown in [Figure number missing]. Figure 5 ,from Figure 5 It can be seen that the separation solution of peak A2 showed better inhibitory effect on angiotensin-converting enzyme (ACE) activity, and the high-performance liquid chromatogram of this peak is shown in [Figure 1]. Figure 6 ,from Figure 6 The liquid chromatography results show a single peak, indicating that purification 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] Experimental Example 2: LC-MS Identification The A2 peak fraction 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 per full scan (MS2 scan); the raw mass spectrometry file was retrieved from the relevant database (Equuscaballus) using Mascot 2.2 software, and finally, the protein identification results were obtained.

[0015] See results Figure 7 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 sequence is WTPYR.

[0016] Experiment Example 3: Molecular docking of the small molecule peptide WTPYR with angiotensin-converting enzyme (ACE) The corresponding three-dimensional model of angiotensin-converting enzyme (PDB number: 2QMJ) was obtained and downloaded from the RCSB Protein Data Bank database. Flexible molecular docking of WTPYR with angiotensin-converting enzyme (ACE) was performed using CB DOCK2. Ligands were extracted from the receptor, water molecules were removed, and polar hydrogen was added. The docking model was screened using its scoring system. Visual analysis of the docking molecular complex determined the key amino acid residues and interaction forces between the complex and angiotensin-converting enzyme (ACE). Results are shown in [Figure number missing]. Figure 8 As shown in the figure, WTPYR interacts with 32 amino acid residues, including hydrogen bonds, hydrophobic bonds, and ionic bonds. WTPYR forms hydrophobic bonds with angiotensin-converting enzyme (ACE) at F391, F287, F527, A354, V380, Y523, F457, T282, W279, and H387; hydrogen bonds at E411, H383, S355, H531, E162, K454, and D415; and ionic bonds at E384, E376, and H353.

[0017] Experimental Example 4: Determination of the inhibitory activity of the small molecule peptide WTPYR on angiotensin-converting enzyme (ACE) The small peptide sequence was synthesized by Shanghai Sangon Biotech Co., Ltd., with a purity ≥95%. The obtained small peptides were serially dissolved to obtain small peptide solutions of 100, 200, 300, 400, and 500 μg / mL. These solutions were then mixed with FAPPG substrate, ACE, and buffer, and the initial absorbance was measured at 340 nm using a microplate reader. The mixture was incubated at 37 ℃ with shaking for 30 min. The absorbance of the reaction mixture was measured again at 340 nm using a microplate reader. The inhibition of angiotensin-converting enzyme (ACE) activity by different concentrations of small peptide solutions was determined by calculating the change in absorbance before and after the reaction. The results are shown in the figure below. Figure 9 The small molecule peptide WTPYR inhibited angiotensin-converting enzyme (ACE) by 89.47% at a concentration of 200 μg / mL.

Claims

1. A bovine whey-derived small molecule peptide with the amino acid sequence Trp-Thr-Pro-Tyr-Arg.

2. The use of the bovine whey-derived small molecule peptide according to claim 1 in the preparation of agents for the prevention and / or treatment of hypertension.

3. The application according to claim 2, characterized in that: Bovine whey protein small molecule peptides have angiotensin-converting enzyme inhibitory activity.

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