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 Ala-Leuu-Pro-Met-Tyr, and the molecular weight of the milk-derived small molecule peptide is 593.731 Da. The small molecule peptide disclosed by the invention has ACE (Angiotensin Converting Enzyme) inhibitory activity, and the highest inhibition rate of the small molecule peptide is 86.17%; the invention has application value in preparation of antihypertensive products, has the characteristics of simple structure, safety, strong activity and the like, 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 an active small molecule peptide derived from whey and its application in the preparation of antihypertensive agents. Background Technology

[0002] Cardiovascular disease is a major threat to human health worldwide, and hypertension, a common cardiovascular disease, is a major risk factor for stroke, myocardial infarction, and other diseases. Angiotensin-converting enzyme (ACE), as a core enzyme of the renin-angiotensin system (RAS) and kallikrein-kinin system (KKS), plays a crucial role in the occurrence and development of hypertension. Therefore, ACE inhibitors are considered one of the best drugs for lowering blood pressure. While traditional ACE inhibitors are widely used in the treatment of hypertension, long-term use can easily cause side effects such as coughing and loss of taste. Therefore, the development of safe and effective food-derived antihypertensive products has become a hot topic.

[0003] Milk, rich in nutrients and readily available, has become a key research focus. Hydrolysis of proteins such as casein in milk can produce peptides with ACE inhibitory activity. Small molecule peptides with ACE inhibitory activity can suppress ACE activity, reduce angiotensin II production, and thus exert a blood pressure-lowering effect, without toxic side effects on the human body, showing broad application prospects. With increasing health awareness and demand for functional foods, developing small molecule peptides with ACE inhibitory activity is of significant practical importance. It can provide a safe and natural blood pressure-lowering option for hypertensive patients and high-risk groups of cardiovascular disease, and also meet consumers' pursuit of health. Summary of the Invention

[0004] This invention provides a small molecule peptide derived from bovine whey fermentation. The amino acid sequence of this small molecule peptide is Ala-Leu-Pro-Met-Tyr (ALPMY), and its molecular weight is 593.731 Da. It has ACE inhibitory activity and can be used to prepare angiotensin-converting enzyme (ACE) inhibitors.

[0005] Another objective of this invention is to apply the above-mentioned milk-derived small molecule peptides in the preparation of antihypertensive drugs.

[0006] The drug of this invention comprises (or contains) the aforementioned small molecule peptides, and may also include one or more pharmaceutical or food-acceptable excipients to improve drug or food absorption or facilitate use, and to formulate suitable dosage forms, such as capsules, pills, powders, tablets, granules, oral liquids, and injections, i.e., pharmaceutically suitable dosage forms or food-appropriate consumption methods; it can be applied to the preparation of functional foods, health products, and drugs for the treatment or adjuvant treatment of obesity or prevention and treatment of hyperlipidemia.

[0007] The objective of this invention is achieved through the following solution: 1. Inoculate the yeast into YPD liquid medium and culture at 20-40℃ and 60-250rpm for 8-46h, then passage 2-3 times for later use; inoculate the lactic acid bacteria into MRS liquid medium and culture at 25-40℃ and 0-150rpm for 20-72h, then passage 2-3 times for later use; the lactic acid bacteria mentioned above are Lactobacillus helveticus and the yeast is Kluyveromyces martensii.

[0008] 2. Preparation of fermented whey Sterilize the bovine whey solution at 90–105°C for 5–10 min. When the temperature drops to room temperature, inoculate with activated Lactobacillus helveticus and Kluyveromyces martensii. The inoculation amount is 1–10% by volume. The fermentation temperature is 20–37°C, the shaking speed is 100–300 rpm, and the fermentation time is 12–50 h. After fermentation, add pepsin to the fermented whey solution. Adjust the pH of the whey solution 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. Centrifuge and collect the supernatant. Freeze-dry to obtain lyophilized fermented whey powder. 3. Dissolve an appropriate amount of lyophilized powder in deionized water to prepare a solution 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 liquid component. Separate and purify the ultrafiltration component with the best inhibitory activity using reverse high-performance liquid chromatography (RP-HPLC). Measure the angiotensin-converting enzyme (ACE) inhibitory activity of the RP-HPLC-purified peak. Then, screen peptides by LC-MS for the peak with the best inhibitory effect on angiotensin-converting enzyme (ACE) activity. Predict the biological activity of the identified peptide sequences using BIOWARE. Subsequently, predict the angiotensin-converting enzyme (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 milk-derived small molecule peptide Ala-Leu-Pro-Met-Tyr (ALPMY) of this invention can significantly inhibit angiotensin-converting enzyme (ACE) activity and can be used to prepare drugs and foods for the prevention and / or treatment of hypertension. It has the advantages of simple preparation, suitability for industrial production, and safety without side effects. Attached Figure Description

[0010] Figure 1The results show the inhibitory activity of solutions containing substances of different molecular weights after ultrafiltration on ACE. Figure 2 A schematic diagram of the separation peaks after RP-HPLC separation of components with molecular weight <3kDa; Figure 3 The results show the ACE 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 separation solution over a time period of 10-15 minutes. Figure 5 The results show the ACE inhibitory activity of the separated solution corresponding to the separation peak after RP-HPLC separation in the 10-15 min time period. Figure 6 The high-performance liquid chromatogram of the separated solution for 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 peptides ALPMY and ACE. Figure 9 The results show the in vitro ACE inhibition activity of the small molecule peptide ALPMY. Detailed Implementation

[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 shall be operated in accordance with conventional methods, and the experimental equipment, reagents and other materials used shall be obtained from commercial sources unless otherwise specified. The angiotensin-converting enzyme inhibition rate was detected using the following methods in the examples below: N-[3-(2-furanyl)acryloyl]-L-phenylalanylglycylglycine (FAPPG) was used as the substrate (3.994 mg FAPPG was added to the matrix buffer, and the volume was adjusted to 10 mL. The mixture was then placed at 4 °C in the dark). 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 the pH of the buffer was adjusted to 8.3 with NaOH solution. The volume was then adjusted to 100 mL for later use). Different concentrations of the small molecule peptide ALPMY (100, 200, 300, 400, 500 μg / mL) were sequentially added to 96-well plates, with the corresponding reagents added according to the reaction system in Table 1. Blank wells were also set up. The absorbance of the reaction system at 340 nm was measured using a microplate reader. The initial absorbance value of the blank well was set as a1, and the initial absorbance value of the sample well was set as b1. The reaction system was incubated in a shaker at 37°C for 30 min, and the absorbance value of the reaction system at 340 nm was measured again using a microplate reader. After the reaction, the absorbance value of the blank well was a2, and the absorbance value of the sample well was b2. The decrease in absorbance value of the blank well was A = a1 - a2, and the decrease in absorbance value of the sample well was B = b1 - b2. The experiment was repeated three times. The angiotensin-converting enzyme inhibition rate was calculated using the following formula. Inhibition rate = 1 - (AB) / A Table 1. Reaction system for ACE inhibitory activity assay ; Experimental 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 and Lactobacillus helveticus into the whey solution at an inoculation ratio of 2% and 4%, respectively. Then ferment at 30℃ and 150 rpm for 48 h. (2) Add 0.3% (w / v) pepsin to the fermented whey solution, adjust the pH of the whey solution after fermentation to 4.30 with 0.1 mol / L hydrochloric acid, shake at 60 rpm and 37 ℃ for 2 h; add 0.1 mol / L sodium hydroxide to adjust the pH to 7.0, add 0.1% (w / v) trypsin and shake at 60 rpm and 37 ℃ for 2 h to simulate enzymatic hydrolysis in vitro, then centrifuge at 4 ℃ and 12000g for 15 min to collect the supernatant, freeze-dry the supernatant to obtain fermented whey freeze-dried powder; (3) Dissolve the lyophilized fermented whey powder from step (2) in deionized water to obtain a 100 mg / mL solution. Filter this component using two ultrafiltration membranes with different cutoff values ​​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 50 mg / mL solution with deionized water. Measure the inhibition rate of each component on ACE activity. The results are shown in […]. Figure 1 ,from Figure 1 It was found that the component with a molecular weight <3kDa had the best inhibitory effect on ACE activity. This component solution was further separated using P-HPLC, and the results are as follows: Figure 2 As shown, according to different time periods Figure 2The peak was divided into five parts. Separates from different time points were collected, and after removing the mobile phase using a rotary evaporator, they were freeze-dried. The freeze-dried components from each time point were then prepared into 10 mg / mL solutions using deionized water. The inhibitory activity of different component solutions on ACE was determined, and the results are shown below. Figure 3 ,from Figure 3 It was found that the fraction with the best inhibitory effect on 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 of 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 ACE was measured. The solution with peak A2 showed the best inhibitory effect on ACE activity. The results are as follows: Figure 5 The high-performance liquid chromatogram of this peak is shown in [reference needed]. Figure 6 ,from Figure 6 The liquid chromatography results show a single peak, indicating that purification is complete.

[0012] The chromatographic conditions for RP-HPLC were as follows: injection volume 1 mL, flow rate 2 L / 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.

[0013] 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 (Equus·caballus) using Mascot 2.2 software, and finally, the protein identification results were obtained.

[0014] See results Figure 7 The corresponding intensity and mass-to-charge ratio of the fragments can be obtained from the figure. Sequence alignment with the database confirms that the small molecule peptide is ALPMY.

[0015] Experimental Example 3: Docking of small molecule peptide ALPMY with ACE molecules The ACE 3D model was downloaded from the RCBSPDB protein database. The 3D structure of small peptides 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, polar hydrogen was added, and the docking model was screened using the Gold score system. Visual analysis of the docked molecular complexes was performed using Moe2019.10 software. The results are shown in [Figure number missing]. Figure 8 As shown in the figure, ALPMY forms one hydrogen bond Q281 with ACE; two hydrophobic bonds A354 and Y523; and one ionic interaction E162, with a total binding energy of -9.1 kcal / mol.

[0016] Example 4: Detection of the inhibitory activity of the small molecule peptide ALPMY against ACE The small peptide sequence was synthesized by Shanghai Sangon Biotech with a purity ≥98%. The obtained small peptide was 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 ACE activity were measured. Figure 9 The small molecule peptide ALPMY inhibited ACE by 86.17% at a concentration of 300 μg / mL.

Claims

1. A milk-derived small molecule peptide with the amino acid sequence Ala-Leu-Pro-Met-Tyr.

2. The use of the milk-derived small molecule peptide according to claim 1 in the preparation of antihypertensive drugs.

3. The application according to claim 2, characterized in that... Milk-derived small molecule peptides have angiotensin-converting enzyme inhibitory activity.

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