Method for preparing ACE inhibitory peptide fermented by marine lactic acid bacteria and application thereof

Abstract

The application discloses a preparation method of marine-source lactic acid bacteria fermentation ACE inhibitory peptide and application thereof, and belongs to the technical field of bioactive peptide and fermentation engineering. 50 The amino acid sequences of the ACE inhibitory peptide are VYPFPGP (SEQ ID NO:1) and PYVPVHF (SEQ ID NO:2). The ACE inhibitory peptide has ACE activity inhibition IC values of 58.46 μM and 9.98 μM respectively. The peptide is obtained by fermenting whey protein by Paracaseolovis casei DS31, and purified by ultrafiltration, gel chromatography and LC-MS / MS identification. The preparation method realizes function-oriented target peptide screening and confirmation, and constructs a green and efficient preparation path which can be amplified, controlled and verified. The application further discloses application of the ACE inhibitory peptide in preparation of a medicine for assisting treatment of hypertension.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides and fermentation engineering technology, specifically to a method for preparing angiotensin-converting enzyme inhibitory peptide derived from marine lactic acid bacteria fermented whey protein and its application. Background Technology

[0002] Hypertension is the leading risk factor for cardiovascular disease worldwide, accounting for over 50% of cardiovascular events. Currently, synthetic angiotensin-converting enzyme (ACE) inhibitors, as one of the mainstream antihypertensive drugs, while possessing significant blood pressure-lowering effects, often limit their long-term clinical application due to adverse reactions such as dry cough and angioedema. Therefore, in recent years, research has gradually shifted its focus to developing safer, edible ACE inhibitory peptides to compensate for the shortcomings of existing synthetic drugs in terms of tolerability and applicability.

[0003] Whey protein, a byproduct of dairy processing, is rich in functional components such as β-lactoglobulin and α-lactalbumin, and contains abundant hydrophobic amino acids, making it an excellent substrate for preparing ACE inhibitory peptides. Its hydrolysates can specifically bind to ACE active sites, inhibiting the conversion of angiotensin I to the vasoconstrictor angiotensin II, thereby exerting a hypotensive effect. Currently, the mainstream technologies for preparing ACE inhibitory peptides from whey protein are enzymatic hydrolysis and microbial fermentation. Among these, microbial fermentation, with its synergistic effect of the strain's own complex protease / peptidase system, continuous reaction, and "biological debittering" advantage, is more likely to yield products with high activity and excellent flavor compared to single enzymatic hydrolysis, and has become a research hotspot in the industry.

[0004] Lactic acid bacteria, Bacillus, yeast, and other microorganisms have all been shown to release ACE-inhibiting peptides in milk systems. However, several technical bottlenecks remain to be overcome in this field: First, existing research often stops at verifying the overall activity of fermentation products, lacking precise separation and targeted extraction of core active peptides, making it difficult to identify the key components responsible for the antihypertensive function, resulting in a lack of product quality control standards. Second, the screened peptides lack systematic multi-dimensional evaluation and characterization, focusing only on in vitro activity indicators, without forming a complete evaluation system encompassing "sequence structure-biological activity-safety-physicochemical properties," leading to insufficient confirmation of product function and hindering their application and transformation in the food or pharmaceutical fields. Third, ACE-inhibiting peptides obtained by existing technologies suffer from insufficient activity, with low half-inhibitory concentrations (IC50). 50 The values ​​are too high, the pressure reduction potential is limited, and it is difficult to meet the needs of practical applications; fourth, the standardization of the preparation process is low, the fermentation conditions and separation and purification parameters lack clear quantitative indicators, and most of them are small-scale laboratory tests. No technical path that can be scaled up has been established. At the same time, there are obvious shortcomings in key industrialization links such as product flavor control and regulatory compliance, which makes it difficult to transform the technological achievements into practical applications. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention proposes the following technical solution:

[0006] In a first aspect, a method for preparing an ACE inhibitory peptide is disclosed, characterized by comprising the following steps:

[0007] S1: Inoculate Lactobacillus casei DS31 seed culture into whey protein solution for fermentation to obtain fermentation broth containing ACE inhibitory peptides.

[0008] In some embodiments, the preparation method further includes S2: centrifuging the fermentation broth to obtain a supernatant, further ultrafiltration treatment, and collecting peptide components with a molecular weight of less than 3 kDa.

[0009] In some embodiments, the preparation method further includes S3: purifying the peptide components by gel chromatography, collecting the elution fractions in groups, measuring the ACE inhibition rate of each group fraction, and obtaining the fraction with the highest ACE inhibition rate;

[0010] S4: The fraction with the highest ACE inhibition rate in S3 was separated and identified by liquid chromatography-tandem mass spectrometry to obtain the ACE inhibitory peptides VYPFPGP or PYVPVHF.

[0011] In some embodiments, the amino acid sequence of the ACE inhibitory peptide is VYPFPGP (as shown in SEQ ID NO:1) or PYVPVHF (as shown in SEQ ID NO:2).

[0012] In some embodiments, the ACE inhibitory peptide with the amino acid sequence SEQ ID NO:1 has an ACE half-inhibition concentration of 58.46 μM.

[0013] In some embodiments, the ACE inhibitory peptide with the amino acid sequence SEQ ID NO:2 has an ACE half-inhibition concentration of 9.98 μM.

[0014] In some embodiments, the ACE inhibitory peptide has a molecular weight range of 757.9-775.9 Da, an isoelectric point range of 5.97-6.3, and a static charge range of 0-0.1.

[0015] In some embodiments, in the fraction with the highest ACE inhibition rate, the mass ratio of the ACE inhibitory peptide with amino acid sequence SEQ ID NO:1 to the ACE inhibitory peptide with amino acid sequence SEQ ID NO:2 is (1~10):(1~10), preferably 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 2:3, 2:5, 2:7, 2:9, 3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 4:1, 4:3, 4:5, 4:7, 4:9, 5:1, 5:2, 5:3, 5:4 The range can be formed by any two of the following values: 5:6, 5:7, 5:8, 5:9, 6:1, 6:5, 6:7, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9, 7:10, 8:1, 8:3, 8:5, 8:7, 8:9, 9:1, 9:2, 9:4, 9:5, 9:7, 9:8, 9:10, 10:1, 10:3, 10:7, 10:9, and any two of the above values.

[0016] In some embodiments, the ACE inhibitory peptide of the amino acid sequence SEQ ID NO:1 has a molecular weight of 775.9 Da, an isoelectric point of 5.97, and a net charge of 0.

[0017] In some embodiments, the ACE inhibitory peptide of the amino acid sequence SEQ ID NO:2 has a molecular weight of 757.9 Da, an isoelectric point of 6.3, and a net charge of +0.1.

[0018] In some embodiments, in step S1, the whey protein solution needs to be sterilized during the preparation process. The sterilization temperature is 100-125°C, and the time is 10-20 minutes. Preferably, the temperature is any one of the following ranges: 100°C, 105°C, 110°C, 115°C, 116°C, 117°C, 118°C, 119°C, 120°C, 121°C, 122°C, 123°C, 124°C, 125°C, or any two of the above values. Preferably, the time is any one of the following ranges: 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, or any two of the above values.

[0019] In some embodiments, in step S1, the concentration of the whey protein solution is 3%-15% (w / v), preferably 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or any two of the above values ​​forming a range.

[0020] In some embodiments, in step S1, the inoculation amount of the Lactobacillus casei DS31 seed solution is 1%-9% (v / v) of the whey protein solution, preferably 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or any two of the above values ​​forming a range.

[0021] In some embodiments, in step S1, the fermentation temperature is 30-42 ℃, preferably 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, or any two of the above values; the rotation speed is 120-220 rpm, preferably 120 rpm, 140 rpm, 180 rpm, 200 rpm, 220 rpm, or any two of the above values; the fermentation time is 24-72 hours, preferably 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours.

[0022] In some embodiments, in step S2, the ultrafiltration process includes first performing pre-filtration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and then performing fine filtration on the permeate using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa.

[0023] In some embodiments, in step S3, the gel chromatography purification uses cross-linked dextran G-15 gel.

[0024] In some embodiments, in step S3, the gel chromatography purification uses deionized water as the mobile phase and elutes at a constant flow rate of 0.3 mL / min.

[0025] Secondly, the use of an ACE inhibitory peptide prepared by the method described in this invention in the preparation of a drug for the adjunctive treatment of hypertension is disclosed.

[0026] In some embodiments, the dosage form of the drug is selected from at least one of fermented milk, solid beverage, compressed candy, gel candy, lyophilized polypeptide powder, capsule, tablet, granule, and oral liquid.

[0027] This invention establishes an in vitro predictive model for screening ACE inhibitory peptides in whey protein breakdown products, comprising the following steps:

[0028] (1) Identify multiple peptide sequences in a mixture of polypeptides obtained from whey protein fermentation and / or enzymatic hydrolysis by LC-MS / MS (liquid chromatography-tandem mass spectrometry);

[0029] (2) Use PeptideRanker to predict the bioactivity of the peptides and screen peptides with a score ≥0.5;

[0030] (3) Three-dimensional structure prediction was performed on peptides with a bioactivity prediction score ≥0.5 using AlphaFold, and energy optimization was performed using Rosetta Relax; human ACE structures were obtained from RCSB PDB, and water molecules were removed and hydrogen atoms were added to the human ACE structures using the molecular visualization software PyMOL; docking was performed using HDOCK, the binding energy of each peptide to ACE was calculated, and peptides with binding energies lower than -50 kcal / mol were selected.

[0031] (4) ToxinPred was used to predict the physicochemical properties and toxicity of peptides with binding energies below -50 kcal / mol.

[0032] This invention employs a multi-level computer-aided screening strategy involving "liquid phase identification, activity prediction, molecular docking, and toxicity assessment," which enables the rapid and precise identification of target peptides with both high activity and high safety from hundreds of peptide fragments. This significantly improves the efficiency and success rate of new peptide discovery while reducing experimental costs.

[0033] This invention uses whey protein as a substrate for fermentation to obtain ACE inhibitory peptides. On the one hand, whey protein is rich in hydrophobic amino acids and various functional protein components beneficial to the human body. Its hydrophobic amino acid side chains help enhance the hydrophobic interaction and coordination stability of the peptide with the ACE active pocket, thereby improving the inhibitory efficacy. On the other hand, whey protein has high purity, which can effectively avoid the interference of non-protein impurities such as polysaccharides on the fermentation and separation process, improving process stability and quality control consistency.

[0034] This invention utilizes *Lactobacillus casei* DS31, a marine-derived strain, for the fermentation of whey protein. This strain carries a complex protease / peptidase system, exhibiting broader cleavage site preferences and environmental tolerance. It can achieve synergistic cleavage of whey protein at multiple sites, preferentially releasing short peptides with smaller molecular weights, higher hydrophobicity, and easier entry into the ACE catalytic cavity. The released short peptides can form multi-site interactions with ACE active sites (such as hydrophobic interactions, hydrogen bonds, and coordination-assisted interactions with zinc ion-related sites), thereby effectively inhibiting its catalytic activity.

[0035] Beneficial effects of the present invention

[0036] 1. Novelty and High Activity of Peptide Sequences: Two novel ACE-inhibiting peptides, VYPFPGP (SEQ ID NO:1) and PYVPVHF (SEQ ID NO:2), were identified for the first time from a system of whey protein fermented by *Lactobacillus casei* DS31 from marine sources. Both short peptides exhibited excellent in vitro ACE-inhibiting activity, with a half-inhibitory concentration (IC50) of [missing value]. 50 The concentrations reached 58.46 μM and 9.98 μM respectively, indicating that it has a high potential for reducing blood pressure.

[0037] 2. Original Innovation and Strain Advantages: The Lactobacillus casei DS31 used in this invention is derived from the marine environment. Its unique complex protease / peptidase system can perform multi-site, synergistic, and efficient hydrolysis of whey protein, preferentially releasing short active peptides with small molecular weight and strong hydrophobicity, overcoming the limitations of traditional single enzymatic hydrolysis products with low activity and single peptide spectrum.

[0038] 3. The screening and characterization system is scientifically rigorous and has a clear objective: This invention constructs a multi-level computer-aided screening strategy of "liquid phase identification - activity prediction - molecular docking - toxicity assessment". This system rapidly and accurately identifies target peptides with both high activity and high safety from a massive number of peptide fragments, greatly improving the efficiency and success rate of new peptide discovery and reducing experimental trial-and-error costs. Simultaneously, the final product undergoes rigorous testing including target peptide concentration determination, ultrafiltration fractionation, gel chromatography purification, LC-MS / MS sequence identification, and in vitro activity verification (ICP-MS). 50 Multi-dimensional characterization methods, including molecular docking simulation and determination, ensured that the structure and function of the target peptide were clearly and reliably confirmed. This systematic screening and confirmation process has not been fully disclosed in previous patent literature in this field.

[0039] 4. The preparation process is green, efficient, and scalable: A complete and scale-up pathway for the targeted preparation of bioactive peptides has been established, integrating microbial fermentation, membrane separation, gel chromatography, and mass spectrometry identification. From strain activation and fermentation condition control to downstream separation, enrichment, and purification, the process design is complete, parameters are clearly defined, and each step is interconnected, forming a complete closed loop from raw materials to high-purity bioactive peptides. The entire process route is simple, primarily using water as a solvent, avoiding the large-scale use of organic solvents, thus meeting the requirements of green production. Furthermore, each unit operation is based on conventional equipment, exhibiting strong scalability and high potential for industrial transformation. Attached Figure Description

[0040] Figure 1 The results show the ACE activity of fermented whey protein from different strains in Example 1 and Comparative Example 1;

[0041] Figure 2 The results show the ACE inhibition rates of different ultrafiltration fractions in Example 3;

[0042] Figure 3 The results of gel chromatography purification of the components with molecular weight <3 kDa in Example 4;

[0043] Figure 4 The results show the ACE inhibition rates of different fractions in Example 4;

[0044] Figure 5 This is a schematic diagram of the docking of the peptide VYPFPGP molecule in Example 6;

[0045] Figure 6 This is a schematic diagram of the docking of the polypeptide PYVPVHF molecule in Example 6;

[0046] Figure 7 IC50 of the two active peptides in Example 6 50 The results are shown in the figures. Figure (a) shows the results for PF-7; Figure (b) shows the results for VP-7; Figure (c) shows the IC50 of the PF-7 peptide. 50 =9.98μM, IC50 of VP-7 peptide 50 =58.46μM.

[0047] Terminology Definition

[0048] In the description of this invention, unless otherwise expressly specified and limited, the following terms shall be understood to have the following meanings:

[0049] 1. ACE Inhibitory Peptides: These are short, bioactive peptides that specifically bind to the active site of angiotensin-converting enzyme (ACE), inhibiting its catalytic conversion of angiotensin I to angiotensin II (a potent vasoconstrictor). They exert their potential antihypertensive effect through this mechanism, and their activity is typically measured at half-inhibitory concentration (IC50). 50 ) characterization.

[0050] 2. Half-inhibitory concentration (IC50) 50 IC50: refers to the concentration of the target peptide required to reduce the catalytic activity of ACE by 50% in an in vitro activity assay system. 50 The smaller the value, the stronger the inhibitory activity of the peptide against ACE.

[0051] 3. Complex protease / peptidase system: refers to the synergistic hydrolysis system carried by Lactobacillus casei DS31 strain, which is composed of multiple proteases (such as endopeptidase and exopeptidase), and can specifically recognize and cleave multiple peptide bonds of whey protein to efficiently release small molecule active peptides.

[0052] 4. Ultrafiltration: refers to the technique of fractionating and separating fermentation supernatant using a semi-permeable membrane with a specific molecular weight cutoff under pressure. In this invention, active peptide components with a molecular weight of less than 3kDa are enriched through two-step ultrafiltration (10 kDa primary filtration → 3 kDa fine filtration).

[0053] 5. Gel chromatography purification: also known as gel filtration chromatography, refers to the technique of separating peptides based on the difference in molecular weight of porous gels such as cross-linked dextran G-15 as the stationary phase. Small peptides can enter the interior of the gel particles and are eluted at a slower rate, while large peptides are excluded from the particles and are eluted at a faster rate, thereby achieving the purpose of purification.

[0054] 6. Sequence identity: refers to the percentage of identical amino acid residues among two polypeptide sequences after alignment. It is calculated using sequence alignment tools (such as BLAST) and is used to assess the homology of polypeptide sequences.

[0055] 7. Computer-aided screening: This refers to a strategy that integrates bioinformatics tools such as PeptideRanker activity prediction, AlphaFold three-dimensional structure modeling, HDOCK molecular docking, and ToxinPred toxicity assessment to screen candidate peptides step by step, aiming to quickly identify target peptides with high activity and low toxicity.

[0056] 8. Whey protein: refers to the water-soluble protein mixture present in whey after milk is precipitated by casein. It is rich in functional components such as β-lactoglobulin and α-lactalbumin, and is a high-quality substrate for preparing ACE inhibitory peptides.

[0057] 9. In vitro activity verification: This refers to the determination of the inhibition rate of the target peptide on ACE catalytic activity by using a colorimetric method with FAPPGG (N-[3-(2-furanyl)acryloyl]-L-phenylalanylglycylglycine) as the substrate under simulated physiological conditions (pH 7.5, 37℃) in vitro. It is a standard in vitro method for evaluating the activity of ACE inhibitory peptides.

[0058] 10. Molecular docking: refers to the method of predicting the interaction (such as hydrophobic interaction, hydrogen bond, coordination interaction) and binding energy between the target peptide and the active pocket of ACE protein through computer simulation technology. The lower the binding energy, the stronger the binding affinity between the peptide and ACE and the higher the potential inhibitory activity. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. However, the implementation of the present invention is not limited thereto, nor is it intended to limit the scope of protection of the present invention.

[0060] The following examples involve Lactobacillus casei ( Lacticaseibacillus casei DS31, deposited on November 28, 2014, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 2, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 10073, was isolated and screened from the intestines of healthy large yellow croaker cultured in net cages in Ningde City, Fujian Province. This strain is publicly protected in patent application CN201510008335.2, entitled "A strain for improving the intestines of aquatic animals and its application."

[0061] Lactobacillus casei ( Lacticaseibacillus caseiThe R-1 strain was deposited on March 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33871. *Lactobacillus casei* R-1, a lactic acid bacterium with good acid resistance and intestinal colonization ability, not only possesses certain health benefits from its fermentation products but also exhibits significant potential for food preservation and shelf-life extension due to the production of abundant antibacterial substances.

[0062] Example 1: Fermentation of ACE inhibitory peptides by Lactobacillus casei DS31

[0063] This embodiment provides a method for preparing angiotensin-converting enzyme (ACE) inhibitory peptides using marine-derived Lactobacillus casei DS31 fermented whey protein. The specific steps are as follows:

[0064] 1. Strain Activation: Marine-derived *Lactobacillus casei* strain DS31 was retrieved from a -80 °C cryopreservation tube. It was streaked onto MRS solid agar plates using a sterile inoculation loop and incubated at 37 °C until single colonies appeared. Typical single colonies were picked and subcultured a second time on the same MRS plates to ensure strain purification and viability. After incubation, 8-10 morphologically sound single colonies were picked using a sterile inoculation loop and inoculated into test tubes containing MRS liquid agar. The tubes were incubated at 37 °C and 180 rpm for 18 hours to obtain a seed culture in the late logarithmic growth phase.

[0065] 2. Preparation of whey protein solution: Weigh whey protein powder, dissolve it in deionized water, stir until fully dissolved, and prepare a whey protein solution with a mass fraction of 11% (w / v). Sterilize at 121°C for 15 minutes. After sterilization, cool the whey protein solution to approximately 37°C.

[0066] 3. Fermentation process: Under aseptic conditions, the prepared seed culture was inoculated into the sterilized and cooled whey protein solution at an inoculation rate of 3% (v / v). The inoculated fermentation system was placed in a constant temperature shaking incubator at 37°C and 180 rpm for 48 hours to obtain the fermentation broth.

[0067] 4. Quantitative analysis of two ACE-inhibiting peptide fragments in the supernatant was performed using the internal standard method:

[0068] (1) Solution preparation:

[0069] a. Internal standard solution: Accurately weigh the chemically synthesized proline isotope-labeled internal standard (Pro-IS) peptide as the internal standard, and prepare an internal standard stock solution with a concentration of 1.00 mg / mL using 0.1% trifluoroacetic acid aqueous solution. Dilute to a working concentration of 0.10 mg / mL before use.

[0070] b. Standard solutions: Accurately weigh the chemically synthesized ACE inhibitory peptide target peptides VYPFPGP and PYVPVHF, and prepare stock solutions with a concentration of 1.00 mg / mL using 0.1% trifluoroacetic acid aqueous solution. Prepare standard solutions of multiple concentrations (0.001, 0.005, 0.01, 0.05 mg / mL) to establish a standard curve, keeping the concentration of the internal standard peptide fixed at 0.1 mg / mL.

[0071] (2) Sample pretreatment: Centrifuge the fermentation broth at 4°C and 8000×g for 15 minutes, filter through a 0.22μm filter membrane, take 100 μL (1mg / ml) of fermentation supernatant, add 10 μL of the above internal standard working solution, and vortex mix evenly.

[0072] (3) Chromatographic analysis conditions: High performance liquid chromatography system, C18 column (2 × 250 mm, 5 μm), column temperature 40 °C, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile solution. Gradient elution program: 0-2 min, maintain 5% B; 2-12 min, B phase linearly increases from 5% to 40%; 12-20 min, B phase linearly increases from 40% to 60%; 20-25 min, B phase linearly increases from 60% to 90% B; 25-29 min, B phase maintains 90%; 29-31 min, B phase decreases to 5% to equilibrate the column. Flow rate: 0.5 mL / min, detection wavelength: 220 nm, injection volume: 10 μL.

[0073] (4) Quantification and calculation: Based on the injection of target peptide solutions of different concentrations, the peak areas of the target peptide and the internal standard peptide are obtained, the peak area ratio is calculated, and an equation is obtained using linear regression to calculate the peak area ratio of the target peptide:

[0074] (Formula I)

[0075] Among them, R sample It is the peak area ratio in the sample, A target_sample A represents the peak area of ​​the target peptide. IS_sample This represents the peak area of ​​the internal standard peptide.

[0076] Calculate the target peptide concentration using the standard curve formula:

[0077] (Formula II)

[0078] Among them, R sample is the peak area ratio in the sample, B is the intercept of the standard curve, and S is the slope of the standard curve.

[0079] The concentrations of ACE inhibitory peptides in the fermentation broth prepared in this embodiment were calculated to be: VYPFPGP concentration of 40 μg / mL and PYVPVHF concentration of 60 μg / mL.

[0080] Comparative Example 1: Fermentation of ACE inhibitory peptides by Lactobacillus casei R-1

[0081] To compare the differences in fermentation effects among different strains, this comparative example used the same fermentation method as Example 1, the only difference being that the strain used was marine-derived Lactobacillus casei (Lactobacillus casei). Lacticaseibacilluscasei The strain R-1, with accession number CGMCC NO.33871.

[0082] Example 2 Preliminary determination of ACE inhibitory activity

[0083] The ACE inhibitory activity of fermented and unfermented whey protein samples was determined by a colorimetric method using FAPPGG (N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine) as a substrate. The specific steps are as follows:

[0084] 1. Reagent preparation

[0085] (1) Tris-HCl-NaCl buffer: 50 mmol / L Tris, 0.3 mol / L NaCl, pH 7.5.

[0086] (2) Substrate working solution: Weigh FAPGG powder and dissolve it in the above Tris-HCl-NaCl buffer solution to prepare a solution with a concentration of 0.1 mmol / L.

[0087] (3) ACE working solution: Take ACE standard and dilute it with Tris-HCl-NaCl buffer (pH 7.5) that has been pre-cooled in an ice bath to prepare a working solution with a concentration of 0.1 U / mL.

[0088] 2. Sample pretreatment

[0089] (1) Preparation of fermentation samples: The fermentation broth obtained in Example 1 was centrifuged at 4°C and 12,000×g for 15 minutes. The supernatant was filtered through a 0.22 μm aqueous microporous membrane. The resulting filtrate was the sample to be tested, denoted as “FWPC-DS31”. The sample to be tested in Comparative Example 1 was obtained by the same method and denoted as “FWPC-R1”.

[0090] (2) Preparation of unfermented control group sample: Take 11% (w / v) whey protein solution with the same composition as in Example 1, which was sterilized at 121°C for 15 minutes but not inoculated with Lactobacillus casei DS31. Under the same pretreatment conditions as the fermentation group (i.e., centrifuged at 4°C, 12,000×g for 15 minutes, and the supernatant was filtered through a 0.22 μm aqueous microporous membrane), the obtained filtrate is the unfermented control group sample to be tested, and is denoted as "CK".

[0091] 3. Measurement Procedure

[0092] Using a 96-well UV plate, the following reaction system and sequence were followed:

[0093] (1) Sample set: Add 50 μL of FAPGG (N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine) substrate working solution to the wells. Add 40 μL of mixture (containing 30 μL buffer and 10 μL of sample to be tested). Quickly add 10 μL of ACE working solution and gently mix several times using a pipette. Immediately place in a microplate reader at 37°C and read the initial absorbance value at 340 nm. After incubating the reaction system precisely at 37°C for 30 minutes, read the absorbance value again.

[0094] (2) Blank control group: Replace “10 μL test sample” in the sample group with “10 μL buffer”, and the rest of the steps are exactly the same as the sample group. This is used to correct the non-enzymatic hydrolysis of the substrate.

[0095] (3) Sample background group: Replace “10 μL ACE enzyme working solution” in the sample group with “10 μL buffer”. The remaining steps are exactly the same as the sample group. This is used to subtract the absorbance interference of the sample itself.

[0096] 4. Activity Calculation

[0097] Based on the absorbance values ​​measured in each group, the ACE inhibition rate was calculated using the following formula:

[0098] Formula III: ΔA_control = A_control(initial) - A_control(30min), which is the decrease in absorbance of the blank control.

[0099] Formula IV: ΔA_inhibitor = A_sample(initial) - A_sample(30min), where ΔA_inhibitor is the decrease in absorbance of the sample group.

[0100] Formula V: ΔA_sample_blank = A_sample_blank(initial) - A_sample_blank(30min), representing the change in absorbance of the background sample group.

[0101] Formula VI:

[0102] ACE inhibition rate (ACE-I (%)) = { 1 - [ (ΔA_inhibitor - ΔA_sample_blank) / ΔA_control ]} × 100%

[0103] The results are as follows Figure 1 As shown, the whey protein solution in the unfermented group exhibited extremely low ACE inhibitory activity, indicating that the inherent ACE inhibitory potential of whey protein that has not undergone microbial transformation is very limited. In contrast, in the fermented group, the ACE inhibition rate of the *Lactobacillus casei* DS31 group reached 86%, significantly higher than that of the unfermented group and the *Lactobacillus casei* R-1 group.

[0104] Example 3: Ultrafiltration Separation and Enrichment of Active Peptide Components

[0105] The fermentation supernatant obtained in Example 1 was fractionated using a plate-scale ultrafiltration device.

[0106] 1. Use an ultrafiltration membrane with a molecular weight cutoff of 10 kDa for initial filtration to obtain components with a molecular weight ≥10 kDa (retentate) and components with a molecular weight <10 kDa (permeate).

[0107] 2. For the above-mentioned components <10 kDa, further filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain components with a molecular weight ≥3 kDa (retentate) and components with a molecular weight <3 kDa (permeate).

[0108] 3. The in vitro ACE inhibitory activity of the above components (≥10 kDa, <10 kDa and ≥3 kDa, <3 kDa) was determined using the same method as in Example 2.

[0109] 4. Results are as follows Figure 2 As shown, the component with a molecular weight <3 kDa exhibited the highest ACE inhibitory activity, reaching 70%. Therefore, the <3 kDa active component was collected, freeze-dried, and stored at -20°C in the dark.

[0110] Example 4: Gel chromatography purification of the target peptide

[0111] (1) Take the polypeptide sample obtained in Example 3 and prepare a solution with a concentration of 448 mg / mL using deionized water.

[0112] (2) Separation was performed using a glass chromatography column packed with Sephadex G-15 gel (specifications: column length 100 cm × inner diameter 1.6 cm). After equilibrating the column with 3 times the column bed volume of deionized water as the mobile phase, 2.0 mL of the above sample solution was loaded onto the column.

[0113] (3) After loading the sample, isocratic elution was performed with deionized water at a constant flow rate of 0.3 mL / min. The entire elution process was monitored in real time at a wavelength of 228 nm using a UV detector. Elution fractions were collected at a rate of one tube every 2 minutes (i.e., 0.6 mL per tube), for a total of 135 tubes.

[0114] (4) After elution, obtain the corresponding elution curve ( Figure 3 Based on the ultraviolet absorption peak shape at 228 nm, the collected fractions were combined into 7 major components (AG), and the in vitro ACE inhibitory activity of the 7 combined components was determined using the method in Example 2.

[0115] The results are as follows Figure 4 As shown, the combined component E (corresponding to collection tube numbers 76-99) exhibited the strongest inhibitory activity, with an ACE inhibition rate of 90%.

[0116] Example 5: Identification of peptide sequences from active fractions

[0117] The most active component E from Example 4 was desalted to remove impurities such as salts that might interfere with mass spectrometry analysis. Specifically, the sample was loaded onto a C18 desalting column that had been activated with acetonitrile solution containing 0.1% trifluoroacetic acid and equilibrated with an aqueous solution containing 0.1% trifluoroacetic acid. Salts were removed by washing with an aqueous solution containing 0.1% trifluoroacetic acid, and finally, the target peptide was eluted with a 30%-50% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid and collected. After vacuum centrifugation and drying, the peptide was redissolved in an appropriate amount of deionized water. The desalted peptide solution was quantified using a UV spectrophotometer at a wavelength of 220 nm to determine its peptide concentration. Based on the peptide quantification results, the solution was then precisely diluted to a concentration of 0.25 μg / μL using mass spectrometry loading buffer (an aqueous solution containing 2% acetonitrile and 0.1% formic acid). Analysis was performed using a nano-flow high-performance liquid chromatography-high-resolution mass spectrometry system under the following conditions:

[0118] 1. Liquid Chromatography Conditions

[0119] Analytical column: uPAC™ High Throughput column (75 μm × 5.5 cm, Thermo, USA)

[0120] Mobile phases: Phase A: an aqueous solution containing 0.1% formic acid; Phase B: an aqueous solution of 80% acetonitrile containing 0.1% formic acid.

[0121] Flow rate: 300 nL / min

[0122] Elution procedure: Linear gradient elution is used, and the specific procedure is shown in Table 1 below:

[0123] Table 1 Gradient elution program

[0124]

[0125] 2. Tandem mass spectrometry analysis conditions:

[0126] Mass spectrometry primary scan range: 380-980 m / z, secondary scan range: 150-2000 m / z, acquisition mode: DDA; Top 100 (selecting the 100 strongest signals from the precursor ions for secondary fragmentation); Primary mass spectrometry resolution: 240,000, AGC target: 500%, maximum injection time: 3 ms, fragmentation mode: HCD; Secondary mass spectrometry resolution: 80,000-100,000, AGC target: standard, maximum injection time: 10 ms, fixed first mass: 150 m / z; Dynamic exclusion time: 12 s.

[0127] A mixture containing 209 peptides was identified using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), and candidate peptide sequences were obtained.

[0128] Example 6 Computer-aided screening of potential ACE-inhibiting peptides

[0129] In this embodiment, bioinformatics methods were used to screen for highly active and low-toxicity ACE inhibitory peptides from the 209 polypeptide sequences identified in Example 4. The specific steps are as follows:

[0130] 1. Sequence-based initial activity screening: Peptide Ranker was used to predict the generalized bioactivity of 209 peptides. This tool outputs an activity probability score from 0 (low activity) to 1 (high activity). A screening threshold of ≥0.5 was set, and 19 peptides were selected for the next round of analysis.

[0131] 2. Structure-based activity prediction and screening:

[0132] The peptides selected in the previous round of screening were subjected to precise molecular docking to predict their binding ability with ACE.

[0133] (1) Peptide structure preparation: Alpha Fold 3 was used to predict the three-dimensional structure of each peptide, and RosettaRelax was used for energy optimization to obtain its stable conformation.

[0134] (2) Receptor protein preparation: The crystal structure of human ACE was obtained from the RCSB PDB database (PDB ID: 1O8A, resolution 2.00 Å, including the N-terminal catalytic domain). Water molecules were removed from the crystal structure using PyMOL software, hydrogen atoms were added at pH 7.4, and the protonation state was optimized.

[0135] (3) Molecular docking: HDOCK was used for docking, and the binding energy of each peptide to ACE was calculated.

[0136] (4) Screening: Select peptides with binding energy below -50 kcal / mol, and two of them will enter the final round of screening.

[0137] 3. Toxicity and physicochemical property prediction: The physicochemical properties and toxicity of the two highly binding active peptides were evaluated using the ToxinPred online tool. The results are shown in Table 2.

[0138] Table 2. Predicted results of toxicity and physicochemical properties of the two peptide segments.

[0139]

[0140] Molecular docking simulations showed that peptides VYPFPGP (SEQ ID NO:1) and PYVPVHF (SEQ ID NO:2) exhibited strong binding affinity to human ACE protein, with predicted binding energies below the screening criterion of -50 kcal / mol. Further PeptideRanker bioactivity prediction scores were 0.879 and 0.649, respectively, both above the screening threshold of 0.5, indicating high potential bioactivity. Toxicity and physicochemical property predictions are shown in Table 2, with key evaluation indicators as follows: "Non-mutagenic" and "Non-toxic" in both mutagenicity (Ames test) and developmental toxicity predictions; predicted water solubility grade of 4, indicating good water solubility; predicted intestinal absorption grade of 3, suggesting potential for oral absorption; and no skin irritation.

[0141] Based on comprehensive bioactivity prediction, molecular docking binding energy, and toxicity assessment results, peptides VYPFPGP (SEQ ID NO:1) and PYVPVHF (SEQ ID NO:2) were identified as high-potential, high-safety target ACE inhibitory peptides, which are the core compounds of this invention.

[0142] Example 7 Verification of the ACE inhibitory activity of the synthetic peptide

[0143] The in vitro ACE inhibitory activity of the target peptides VYPFPGP (SEQ ID NO:1) and PYVPVHF (SEQ ID NO:2), selected by computer screening, was verified through the following steps:

[0144] 1. Peptide synthesis: Shanghai Jier Biochemical Co., Ltd. was commissioned to synthesize the target peptides VYPFPGP and PYVPVHF using a standard solid-phase synthesis method. The peptides were then purified by high performance liquid chromatography (HPLC), with a purity of >95%.

[0145] 2. Sample preparation: Prepare a series of solutions of known concentrations using deionized water to synthesize the synthetic peptide.

[0146] 3. Activity assay: The ACE inhibition rate of the two synthetic peptides at different concentrations was measured strictly according to the in vitro ACE inhibition activity assay method described in Example 2.

[0147] 4. Data Processing: Based on the measurement results, a dose-inhibition rate curve was plotted, and the half-inhibitory concentration (IC50) of each synthetic peptide was calculated. 50 The values ​​are: VYPFPGP (SEQ ID NO:1) is denoted as “VP-7”, and PYVPVHF (SEQ ID NO:2) is denoted as “PF-7”.

[0148] like Figure 5-6 This diagram illustrates the molecular docking of two peptide segments, demonstrating the strong affinity between the peptide and ACE. Figure 7 As shown, the chemically synthesized peptides VYPFPGP (SEQ ID NO:1) and PYVPVHF (SEQ ID NO:2) both exhibited significant ACE inhibitory activity. Their IC50 values... 50 The values ​​were 58.46 μM and 9.98 μM, respectively. This result directly confirms that the peptides with these two specific amino acid sequences are the effective substances for ACE inhibition in this invention.

[0149] This invention constructs a highly efficient and reproducible method for preparing ACE-inhibiting peptides by systematically optimizing key process parameters such as sterilization temperature, substrate addition amount, inoculum size, and fermentation time. The peptides obtained using this method exhibit excellent ACE-inhibiting activity. The method disclosed in this invention is simple, has a low operating threshold, and allows for quantifiable parameters, facilitating scale-up from small-scale to pilot-scale industrial production. It is compatible with conventional downstream processes such as membrane separation and gel filtration, and possesses good industrial adaptability and cost controllability.

[0150] It should be noted that the above embodiments are used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Those skilled in the art can make equivalent substitutions or optimizations to process parameters (such as temperature, pH, inoculum size, fermentation time), separation strategies (membrane molecular weight cutoff, elution conditions), and strain sources (strains of the same genus or equivalent enzyme-producing spectrum) without departing from the essence of the present invention and the scope of protection of the claims; all such substitutions or optimizations should be covered within the scope of protection of the present invention.

[0151] This specification describes embodiments for ease of understanding, but it does not mean that each embodiment contains only a single independent technical solution. The specification should be understood as a whole, and the technical features in each embodiment can be reasonably combined and reconfigured without conflict to form other equivalent technical solutions that can be implemented by those skilled in the art.

Claims

1. The use of ACE inhibitory peptides in the preparation of antihypertensive drugs, characterized in that, The amino acid sequence of the ACE inhibitory peptide is PYVPVHF.

2. The use according to claim 1, characterized in that, The dosage form of the drug is selected from at least one of the following: polypeptide lyophilized powder, capsules, tablets, granules, and oral liquid.

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