A method for preparing corn peptide for reducing blood pressure and application thereof

Abstract

This invention relates to the field of functional food processing technology, and discloses a method for preparing and applying corn peptides that lower blood pressure. First, microwave pretreatment is used to improve the dissolution rate of corn protein. Then, dynamic temperature-controlled synergistic enzymatic hydrolysis combined with time-segmented microwave assistance is used to directionally release hydrophobic ACE-inhibiting peptides. Surface-modified magnetic materials are used to perform biomimetic affinity adsorption and magnetic separation purification of the target peptides, achieving highly selective enrichment of active ingredients. Finally, the peptides are compounded with stabilizers such as tea polyphenol-zinc chelate and Monascus purpureus extract to form a charge-matched micelle protection system. Through dynamic temperature-controlled synergistic enzymatic hydrolysis and biomimetic magnetic separation processes, efficient release and functional enhancement of active peptides are achieved, simultaneously improving ACE inhibitory activity and storage stability. The charge-matching of stabilizers and the synergistic design of components overcome the absorption barrier of active peptides. A multi-pathway regulatory system is constructed, integrating "blood pressure lowering, lipid lowering, and antioxidant" functions. Experiments have demonstrated that this application has a blood pressure lowering effect.

Description

Technical Field

[0001] This invention relates to the field of functional food processing technology, specifically to a method for preparing and applying corn peptides that lower blood pressure. Background Technology

[0002] Corn peptides, as an important category of plant-derived bioactive peptides, have become a research hotspot in the field of functional foods in recent years due to their significant ACE inhibitory activity and potential blood pressure-lowering function. However, traditional preparation processes often rely on a single enzymatic hydrolysis temperature or isothermal conditions, which are difficult to adapt to the dynamic conformational changes required by multi-enzyme systems, resulting in low enzymatic hydrolysis efficiency and insufficient release of hydrophobic active peptides. Existing separation and purification techniques (such as ultrafiltration and ion exchange chromatography) often cause loss of target peptides and destruction of active conformations due to non-specific adsorption or mechanical shearing. At the same time, there is a lack of targeted enrichment methods for ACE-inhibiting peptides, resulting in a low proportion of effective components in the final product.

[0003] Regarding stability control, conventional processes do not fully consider the risks of oxidative degradation and enzymatic inactivation of bioactive peptides during storage and digestion. In particular, hydrophobic peptides are prone to decreased bioavailability due to aggregation or phase transition. Furthermore, existing technologies mostly focus on the preparation of single active ingredients and lack functional design for multi-pathway synergistic regulation (such as combined blood pressure and blood lipid management), which limits the application scenarios and market competitiveness of products. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying corn peptides that lower blood pressure, solving the problems of low release efficiency of active peptides, poor separation selectivity, insufficient stability, and limited functionality in traditional corn peptide preparation processes.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing corn peptides that lower blood pressure, comprising the following steps: S1. Raw material pretreatment: Corn protein is extracted by alkali dissolution and acid precipitation, and then subjected to microwave pretreatment; The raw material pretreatment employs an alkali-soluble acid-precipitation method to extract zein, followed by microwave pulse treatment to disrupt its β-sheet hydrophobic structure. The non-thermal effects of microwaves (such as high-frequency electromagnetic field vibration) selectively disrupt the rigid regions of the protein, exposing enzymatic sites and providing more readily attackable substrate conformations for subsequent multi-stage enzymatic hydrolysis. This step, combining physical pretreatment with chemical extraction, overcomes the bottleneck of enzymatic hydrolysis efficiency caused by the low solubility of zein in traditional processes.

[0006] S2, Dynamic Temperature Synergistic Enzymatic Hydrolysis: The pretreated corn protein is subjected to three stages of enzymatic hydrolysis: low temperature, high temperature, and low temperature. Different proteases are added in each stage, and microwave assistance is applied at different times. Dynamic temperature-dependent enzymatic hydrolysis is divided into three stages: low temperature, high temperature, and low temperature, corresponding to the activity optimization ranges of alkaline protease, chymotrypsin, and glutaminase, respectively. First stage (low temperature): At 43-47℃, the hydrophilic cleavage ability of alkaline protease is activated, and pulsed microwaves are applied simultaneously to accelerate the breakage of macromolecular chains through mechanical shearing effect. The second stage (high temperature): The temperature is raised to 53-57℃ to trigger the allosteric effect of chymotrypsin, which enhances its recognition and cleavage of hydrophobic peptides. The third stage (low temperature): The temperature is lowered to 33-37℃ to induce hydrogen bond anchoring between glutaminase and hydrophobic peptides. In the final stage, low-power microwaves are applied to assist in the release of the target peptides.

[0007] Temperature-enzyme conformation association design: By varying the temperature to regulate the conformation of the active sites of different enzymes (such as high temperature inducing the opening of the hydrophobic pocket of chymotrypsin), multi-enzyme synergistic directional cleavage can be achieved.

[0008] Timing microwave coupling: Microwave input is controlled in stages (high power for initial disruption, low power for final release) to avoid enzyme inactivation caused by microwaves throughout the process and improve energy utilization efficiency.

[0009] S3. In-situ magnetic separation and purification: Surface-modified magnetic materials are added to the enzymatic hydrolysate to adsorb the target peptide fragments, followed by magnetic separation and elution. Surface-modified aminated Fe3O4@SiO2 magnetic microspheres were directly added to the enzymatic hydrolysate, with ACE-inhibiting peptide affinity ligands (such as Captopril analogs) grafted onto their surfaces. The magnetic material selectively adsorbed target peptides with molecular weight less than 1 kDa and ACE-inhibiting activity by sieving through pore size and specifically binding to the ligand-peptide combination. Subsequently, the peptides were separated by magnetic field and eluted with acidic buffer.

[0010] Bionic affinity adsorption: Affinity ligands mimic the structure of the active site of ACE enzymes, preferentially capturing peptides with depressurization potential through hydrogen bonding and hydrophobic interactions, overcoming the limitations of traditional separation techniques that rely solely on molecular weight differences.

[0011] In-situ separation and loss reduction: The target product is simultaneously adsorbed during the enzymatic hydrolysis process, avoiding the loss of activity caused by high temperature or extreme pH treatment after the reaction is terminated.

[0012] S4. Stabilizer compounding: The purified corn peptides are mixed with stabilizers and dried to obtain the final product.

[0013] The purified corn peptides were compounded with sodium alginate, L-arginine, tea polyphenol-zinc chelate, and Monascus purpureus extract to form a charge-matched complex micelle system. The negatively charged backbone of sodium alginate and the positively charged groups of L-arginine form stable micelles through electrostatic interactions, and their surface zeta potential matches the charge of the intestinal mucosa. The tea polyphenol-zinc chelate stabilizes the α-helical conformation of the peptides through coordination bonds, and MonacolinK from Monascus purpureus extract synergistically inhibits ACE activity with the corn peptides.

[0014] Active transmembrane transport design: The charge properties of the composite micelles enhance the affinity of peptides for the intestinal epithelial cell oligopeptide transporter (PepT1), promoting intestinal absorption.

[0015] Synergistic effect of multiple components: Natural stabilizers not only provide physical protection, but also broaden the antihypertensive pathway through the synergistic effect of bioactive ingredients (such as MonacolinK) and corn peptides.

[0016] Preferably, the dynamic temperature-controlled synergistic enzymatic hydrolysis in step S2 specifically includes: First stage: At 43-47℃, add alkaline protease at an enzyme-to-protein ratio of 1:40-1:60 (w / w) and simultaneously apply pulsed microwaves; Second stage: Heat to 53-57℃, add chymotrypsin at an enzyme-to-protein ratio of 1:80-1:120 (w / w), and turn off the microwave. Third stage: Cool down to 33-37℃, add glutaminase, enzyme-to-protein ratio is 1:70-1:90 (w / w), and apply low-power microwave in the final stage.

[0017] Preferably, the microwave assistance in step S2 satisfies the following conditions: The microwave power in the first stage is 180-220W, the pulse period is 8-12 seconds, and the action time is ≤5 minutes. The third stage uses a microwave power of 100-150W, a pulse period of 5-8 seconds, and an action time of 8-12 minutes.

[0018] Preferably, in step S3, the magnetic material is aminated Fe3O4@SiO2 microspheres with ACE inhibitory peptide affinity ligands grafted onto their surface, wherein the affinity ligands are mercaptopropionylglycine or Captopril analogs.

[0019] Preferably, the three stages of enzymatic hydrolysis in step S2 are as follows: The first stage lasts 25-35 minutes, the second stage lasts 50-70 minutes, and the third stage lasts 25-35 minutes.

[0020] Preferably, in step S3, the amount of magnetic material added is 4-6% (w / w) of the total mass of the enzymatic hydrolysate, and the elution buffer used in the elution process is a glycine-HCl solution with a pH of 2.5-3.5.

[0021] Preferably, the stabilizer in step S4 comprises the following components by mass parts: Sodium alginate 0.3-0.7 parts, L-arginine 0.7-1.3 parts, tea polyphenol-zinc chelate 0.1-0.3 parts, Monascus purpureus extract 0.05-0.15 parts.

[0022] Preferably, the Monascus purpureus extract is prepared by the following steps: Mix the solid fermentation product of Monascus purpureus with 50-70% ethanol solution at a material-to-liquid ratio of 1:10-1:20 (w / v) and extract at 50-60℃ for 1-2 hours. After filtration, the extract is concentrated to 10-20% of its original volume to obtain an extract with a MonacolinK mass percentage of ≥0.2%.

[0023] The present invention also provides the application of a blood pressure-lowering corn peptide prepared by the above preparation method in the preparation of blood pressure-lowering functional foods, wherein the functional foods are solid beverages, compressed candies or dairy products.

[0024] The corn peptide composition prepared by this method can be directly applied to food carriers such as solid beverages and compressed candies. Through charge matching and conformational protection of the stabilizer system, the composition can maintain ACE inhibitory activity under conventional food processing conditions (such as high-temperature sterilization and acidic environments), achieving oral efficacy without relying on nano-encapsulation technology.

[0025] This invention provides a method for preparing corn peptides that lower blood pressure and their applications. It has the following beneficial effects: 1. This invention overcomes the technical bottleneck of low efficiency in traditional single-temperature enzymatic hydrolysis by using a synergistic design of dynamic temperature-controlled enzymatic hydrolysis and staged microwave-assisted hydrolysis. The temperature gradient is adapted to the optimal conformation of different proteases, and the non-thermal effect of microwaves is used to directionally destroy the hydrophobic core of the protein, thereby promoting the efficient release of hydrophobic small molecule peptides with ACE inhibitory activity and significantly improving the targeted biological activity of the product.

[0026] 2. This invention is based on a biomimetic affinity ligand magnetic separation process, which selectively enriches active peptides through molecular recognition mechanisms, avoiding non-specific losses in conventional chromatography or ultrafiltration processes. At the same time, the compounded stabilizer forms a physical-chemical dual protective barrier through charge matching and chelation antioxidant synergistic effects, effectively inhibiting the degradation of active ingredients and ensuring the long-term stability of the end product.

[0027] 3. The present invention optimizes the charge properties of the hydrophobic peptides enriched by magnetic separation with those of the stabilizer micelles, thereby enhancing the solubility and mucosal permeability of the active peptides in the gastrointestinal environment. The amphiphilic peptides are efficiently absorbed by the intestines through a dual pathway of passive diffusion and active transport, overcoming the problem of low bioavailability caused by conformational damage in traditional processes.

[0028] 4. This invention introduces Monascus purpureus extract and tea polyphenol-zinc chelate, achieving three-dimensional regulation of cardiovascular health function through a multi-target synergistic mechanism of "ACE inhibition-lipid metabolism regulation-oxidative stress inhibition". This design breaks through the limitations of the action range of single active ingredients, providing a more comprehensive solution for the development of functional foods. Attached Figure Description

[0029] Figure 1 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Please see the appendix Figure 1 This invention provides a method for preparing and applying a blood pressure-lowering corn peptide through multiple embodiments, the details of which are as follows: Example 1: Raw material pretreatment: Take 100g of corn syrup, add 1.0L of 0.1M NaOH solution (solid-liquid ratio 1:10 w / v), adjust the pH to 10.5, and stir at 50℃ for 45 minutes (300rpm). Centrifuge (10000×g, 18 minutes) to collect the supernatant, adjust the pH to 4.5 with 0.2M HCl, let stand for 1.5 hours, wash the precipitate with water to pH 7.0, and freeze dry to obtain zein (87% purity). The protein powder was prepared into a 10% w / v suspension and treated in a microwave reactor at 200W power, 2450MHz frequency, 10-second pulse cycle for 5 minutes at a temperature ≤50℃.

[0032] Dynamic temperature-dependent enzymatic hydrolysis: First stage: Adjust pH to 8.5, add alkaline protease (enzyme-to-protein ratio 1:50 w / w), react at 45℃ for 30 minutes, and apply 200W pulsed microwave (pulse period 10 seconds) for the first 5 minutes. Second stage: Heat to 55℃, adjust pH to 8.0, add chymotrypsin (enzyme-to-protein ratio 1:100w / w), react for 60 minutes, then turn off the microwave; Third stage: Cool down to 35℃, adjust pH to 6.5, add glutaminase (enzyme-to-base ratio 1:80w / w), react for 30 minutes, and apply 125W pulsed microwave (pulse period 6 seconds) for the last 10 minutes.

[0033] In-situ magnetic separation: Aminated Fe3O4@SiO2 microspheres (surface grafted with Captopril analogue, 5% w / w) were added to the enzymatic hydrolysate and stirred at 30°C for 30 minutes (200 rpm). Microspheres were collected by magnetic separation (magnetic field strength 1.0T) and the target peptide was eluted with pH 3.0 0.1M glycine-HCl buffer.

[0034] Stabilizer compound: The purified corn peptide solution was mixed with the following stabilizers: Sodium alginate 0.5% w / w; L-arginine 1.0% w / w; Tea polyphenol-zinc chelate 0.2% w / w (tea polyphenol:ZnSO4·7H2O=4:1); Monascus purpureus extract 0.1% w / w (from Monascus purpureus solid fermentation product at a material-to-liquid ratio of 1:15 w / v, 60% ethanol, 55℃ for 1.5 hours, and concentrated to 15% of the original volume); Stir the mixture at 45°C for 45 minutes, centrifuge to degas (6500×g, 12 minutes), and spray dry (inlet air 180°C, outlet air 80°C).

[0035] Example 2: Raw material pretreatment: Corn yellow pulp was prepared with 0.05M NaOH solution (solid-liquid ratio 1:15 w / v), pH 10.0, and stirred at 45℃ for 60 minutes. Centrifuge (8000×g, 20 minutes), acid precipitation to pH 4.0, let stand for 2 hours, and wash with water until pH 6.8; Protein suspension concentration 8% w / v, microwave treatment at 180W power, 8-second pulse cycle, for 6 minutes, temperature ≤50℃.

[0036] Dynamic temperature-dependent enzymatic hydrolysis: First stage: pH 8.0, alkaline protease (enzyme-to-protein ratio 1:40 w / w), reaction at 43℃ for 35 minutes, with 180W pulsed microwave applied for the first 5 minutes (pulse period 8 seconds). Second stage: Heat to 53℃, pH 7.5, add chymotrypsin (enzyme-to-protein ratio 1:80w / w), react for 70 minutes; Third stage: Cool down to 33℃, pH 6.0, add glutaminase (enzyme-to-base ratio 1:70w / w), react for 35 minutes, and apply 100W pulsed microwave (pulse period 5 seconds) for the last 12 minutes.

[0037] In-situ magnetic separation: Magnetic microspheres were added at 4% w / w, and stirred at 25°C for 35 minutes. Elution buffer pH 2.5 0.05M glycine-HCl.

[0038] Stabilizer compound: Sodium alginate 0.3% w / w, L-arginine 0.7% w / w, tea polyphenol-zinc 0.1% w / w (3:1), Monascus purpureus extract 0.05% w / w (solid-liquid ratio 1:20 w / v, 50% ethanol, 50°C for 2 hours, concentrated to 20% volume).

[0039] Example 3: Raw material pretreatment: Corn yellow pulp was prepared with 0.15M NaOH solution (solid-liquid ratio 1:10 w / v), pH 11.0, and stirred at 55℃ for 30 minutes. Centrifuge (12000×g, 15 minutes), acid precipitation to pH 5.0, let stand for 1 hour, and wash with water until pH 7.2; Protein suspension concentration 12% w / v, microwave treatment 220W power, 12-second pulse cycle, treatment for 4 minutes, temperature ≤50℃.

[0040] Dynamic temperature-dependent enzymatic hydrolysis: First stage: pH 9.0, alkaline protease (enzyme-to-protein ratio 1:60 w / w), reaction at 47℃ for 25 minutes, with 220W pulsed microwave applied for the first 5 minutes (pulse period 12 seconds). Second stage: Heat to 57℃, pH 8.5, add chymotrypsin (enzyme-to-protein ratio 1:120w / w), react for 50 minutes; Third stage: Cool down to 37℃, pH 7.0, add glutaminase (enzyme-to-base ratio 1:90w / w), react for 25 minutes, and apply 150W pulsed microwave (pulse period 8 seconds) for the last 8 minutes.

[0041] In-situ magnetic separation: Magnetic microspheres were added at 6% w / w, and stirred at 35°C for 25 minutes. Elution buffer pH 3.5 0.15M glycine-HCl.

[0042] Stabilizer compound: Sodium alginate 0.7% w / w, L-arginine 1.3% w / w, tea polyphenol-zinc 0.3% w / w (5:1), Monascus purpureus extract 0.15% w / w (solid-liquid ratio 1:10 w / v, 70% ethanol, 60°C for 1 hour, concentrated to 10% volume).

[0043] Comparative Example 1: Compared with Example 1, the difference is that the microwave preprocessing in step S1 and the microwave assistance in all stages of step S2 are cancelled, while the remaining steps and parameters are the same.

[0044] Comparative Example 2: Compared with Example 1, the difference is that the dynamic temperature-changing enzymatic hydrolysis in step S2 is changed to constant temperature enzymatic hydrolysis (45°C throughout), while the other steps and parameters are the same.

[0045] Comparative Example 3: Compared with Example 1, the difference is that unmodified aminated Fe3O4@SiO2 microspheres (without ACE inhibitory peptide affinity ligands) are used in step S3, while the other steps and parameters are the same.

[0046] Comparative Example 4: Compared with Example 1, the difference is that the stabilizer in step S4 contains only sodium alginate (0.5% w / w) and L-arginine (1.0% w / w), and no tea polyphenol-zinc chelate and Monascus purpureus extract are added. All other steps and parameters are the same.

[0047] Comparative Example 5: Compared with Example 2, the difference is that Monascus extract is not added to the stabilizer in step S4, while the other steps and parameters are the same.

[0048] Comparative Example 6: Compared with Example 2, the difference is that the elution buffer in step S3 is changed to pH 7.0 phosphate buffer, while the other steps and parameters are the same. Comparative Example 7: Compared with Example 3, the difference is that low-power microwaves are not applied during the third stage of enzymatic hydrolysis in step S2, while the remaining steps and parameters are the same.

[0049] Comparative Example 8: Compared with Example 3, the difference is that ultrafiltration (3kDa membrane) is used instead of magnetic separation in step S3, while the other steps and parameters are the same.

[0050] Test Example 1: Comparative groups: Example 1, Comparative Example 1 (without microwave), Comparative Example 2 (isothermal enzymatic hydrolysis); Experimental steps: Sample preparation: Corn peptide samples were prepared according to the methods described in Example 1, Comparative Example 1, and Comparative Example 2, with three batches prepared in parallel for each example.

[0051] ACE inhibition rate assay: Reaction system: Mix 0.1 mL of sample solution (1 mg / mL) with 0.2 mL of ACE enzyme solution (0.1 U / mL, pH 8.3 borate buffer) and pre-incubate at 37°C for 5 minutes; Add 0.2 mL of substrate HHL (5 mM, containing 0.3 M NaCl), and react at 37 °C for 30 minutes; The reaction was terminated by adding 0.5 mL of 1 M HCl.

[0052] Hippuric acid test: The reaction solution was filtered through a 0.45 μm filter membrane, and the amount of hippuric acid produced was determined by HPLC (C18 column, mobile phase methanol-water = 60:40, flow rate 1.0 mL / min, detection at 228 nm).

[0053] Calculate the inhibition rate: Inhibition rate (%) = [1 - (peak area of ​​hippuric acid in sample group / peak area of ​​blank control group)] × 100%.

[0054] Peptide yield determination: Lowry method: Centrifuge the supernatant of the enzymatic hydrolysate (10,000×g, 10 minutes), dilute to a suitable concentration, add Folin-Ciocalteu reagent, measure the absorbance at 725nm, and calculate the soluble peptide content using the casein standard curve.

[0055] Yield calculation: Peptide yield (%) = (mass of soluble peptides / initial protein mass) × 100%.

[0056] Analysis of the proportion of hydrophobic peptides: HPLC-MS method: Take the purified peptide solution (1 mg / mL) and separate it using a C18 column (mobile phase A: 0.1% formic acid water; B: 0.1% formic acid acetonitrile, gradient elution 0-60% B, 30 min). Mass spectrometry data were acquired in ESI positive ion mode, and peptide sequences were analyzed using PeaksStudio software to determine the proportion of hydrophobic amino acids (Val, Leu, Ile, Pro, Phe).

[0057] Experimental data: Table 1. Effects of the synergistic effect of dynamic temperature variation and microwave on the activity of maize peptides. Key points of data analysis: Synergistic effect of microwave and temperature variation: The ACE inhibition rate of Example 1 (~82%) was significantly higher than that of Comparative Example 1 (~51%) and Comparative Example 2 (~63%), indicating that microwave pretreatment and dynamic temperature variation have a synergistic effect on the release of active peptides.

[0058] Peptide yield and hydrophobicity correlation: The proportion of hydrophobic peptides in Example 1 (~43%) was much higher than that in Comparative Example 1 (~23%), proving that microwave assistance can effectively destroy the hydrophobic core of corn protein and release more hydrophobic active peptides.

[0059] Limitations of isothermal enzymatic hydrolysis: The peptide yield of Comparative Example 2 (isothermal 45°C) (~56%) was lower than that of Example 1 (~67%), and the proportion of hydrophobic peptides decreased, indicating that a single temperature cannot activate the optimal conformation of multiple enzymes.

[0060] Experimental Summary: This invention significantly improves the ACE inhibitory activity of corn peptides and the targeted release efficiency of hydrophobic active peptides through the coupling effect of dynamic temperature-assisted enzymatic hydrolysis and microwave assistance. Experimental data show that using a three-stage temperature program (low temperature-high temperature-low temperature) combined with time-segmented microwave input (Example 1) increases the ACE inhibition rate by approximately 20% compared to isothermal enzymatic hydrolysis (Comparative Example 2), and increases the proportion of hydrophobic peptides by nearly 10 percentage points. This result verifies the regulatory effect of temperature gradient changes on protease conformation: in the low temperature stage (43-47℃), the hydrophilic catalytic domain of the alkaline protease is fully exposed, and the mechanical shearing effect of pulsed microwaves accelerates the depolymerization of the hydrophobic core; in the high temperature stage (53-57℃), the hydrophobic pocket of chymotrypsin is induced to open, enhancing the cleavage efficiency of internal peptide bonds; in the final low temperature stage (33-37℃), the target peptide is stabilized by the hydrogen bond anchoring effect of glutaminase, while low-power microwave assistance promotes the release of hydrophobic peptides from the enzymatic hydrolysis complex.

[0061] The synergistic design of timing control of microwave energy and variable temperature programs is key to overcoming the bottlenecks of traditional enzymatic hydrolysis processes. In Comparative Example 1 (without microwave), the ACE inhibition rate decreased to approximately 50%, and the proportion of hydrophobic peptides was less than 25%, indicating that microwave treatment not only accelerates the mass transfer process, but its non-thermal effects (such as protein molecular polarization induced by high-frequency electric fields) can also directionally disrupt the β-sheet structure, exposing more cleavage sites. In contrast, isothermal enzymatic hydrolysis (Comparative Example 2) suffers from limited hydrolysis efficiency and a significantly reduced amount of hydrophobic peptides due to its inability to adapt to the optimal conformation of multiple enzymes.

[0062] The synergistic effect of dynamic temperature variation and microwave input is further reflected in the structure-activity relationship of the product. In Example 1, the proportion of hydrophobic peptides is as high as 43%. These peptide chains, rich in hydrophobic amino acids such as Val and Pro, can tightly bind to the active pockets (e.g., the S2' hydrophobic cavity) of ACE enzymes through hydrophobic interactions, thereby enhancing inhibitory activity. In contrast, Comparative Examples 1-2, lacking the aforementioned synergistic mechanism, produced peptides predominantly composed of hydrophilic components, making it difficult to effectively block ACE catalytic sites. This result confirms the innovation of this invention in synergistically regulating the conformation of active peptides through a physical field-enzymatic reaction, providing a reliable pathway for the industrial preparation of highly active antihypertensive peptides.

[0063] Test Example 2: Comparative groups: Example 1, Comparative Example 3 (unmodified magnetic material), Comparative Example 4 (without tea polyphenols-zinc and Monascus purpureus).

[0064] Experimental steps: Sample preparation: Corn peptide compositions were prepared according to the methods described in Example 1, Comparative Example 3, and Comparative Example 4, with three batches prepared in parallel for each example.

[0065] Determination of target peptide recovery rate: BCA method for determining peptide content: Take the enzymatic hydrolysate before and after magnetic separation (dilute to the linear range), mix with BCA reagent (Sigma, BCA1), incubate at 60°C for 30 minutes, measure the absorbance at 562 nm, and calculate the peptide concentration using the bovine serum albumin (BSA) standard curve.

[0066] Recovery rate calculation: Recovery rate (%) = (mass of peptides in eluent / mass of peptides in initial enzymatic hydrolysate) × 100%.

[0067] Storage stability test: Sample processing: The corn peptide composition was dispensed into transparent glass bottles and stored at 4°C and 25°C in the dark, respectively. Samples were taken for testing on days 0, 15, and 30.

[0068] ACE inhibitory activity retention rate: The ACE inhibition rate was determined according to the method in Test Example 1, and the retention rate was calculated as (inhibition rate after storage / initial inhibition rate) × 100%.

[0069] Detection of Monacolin K content in Monascus purpureus extract: HPLC detection conditions: C18 column (4.6×250mm, 5μm), mobile phase acetonitrile-0.1% phosphoric acid aqueous solution (65:35), flow rate 1.0mL / min, detection wavelength 237nm.

[0070] Standard curve method: A concentration-peak area standard curve was established using Monacolin K standard (Sigma, 79880), and the content of Monacolin K in the extract was calculated using the external standard method.

[0071] Experimental data: Table 2. Effects of magnetic separation and stabilizer combination on the properties of corn peptides Key points of data analysis: The key role of magnetic material affinity ligands: The recovery rate of the target peptide in Example 1 (~88%) was significantly higher than that in Comparative Example 3 (~54%), indicating that the surface-modified Captopril analog selectively captures ACE-inhibiting peptides through biomimetic affinity adsorption.

[0072] Synergistic protective effect of stabilizers: After storage at 25°C for 30 days, the ACE activity retention rate of Comparative Example 4 (without tea polyphenols-zinc and Monascus purpureus) decreased to about 56%, while the retention rate of Example 1 was still 85%, which proves that the antioxidant effect of tea polyphenols-zinc and the synergistic effect of Monascus purpureus extract are crucial to stability.

[0073] Quantitative control of Monacolin K: In Example 1, the Monacolin K content of the Monascus purpureus extract was consistently above 2.1 mg / g, meeting the requirement of ≥0.2% mass percentage in the claims.

[0074] Experimental Summary: The magnetic separation process of this invention achieves selective enrichment of highly active peptides through biomimetic affinity ligand design. Experimental data show that the target peptide recovery rate (~88%) of magnetic microspheres grafted with Captopril analogs (Example 1) is significantly higher than that of unmodified materials (Comparative Example 3, ~54%). This difference stems from the specific binding mechanism of affinity ligands to ACE inhibitory peptides: the thiol and carboxylic acid groups of Captopril analogs can mimic the zinc ion binding domain of the ACE enzyme active site, preferentially capturing peptides containing key sequences such as Pro-Val through hydrogen bonding and hydrophobic interactions. In contrast, unmodified magnetic materials rely solely on pore size sieving, failing to distinguish between active and inactive components, resulting in the non-specific adsorption of a large number of non-target peptides.

[0075] The stabilizer compound system ensures the storage stability of the product through a dual mechanism of charge matching and activity synergy. In Example 1, after 30 days of storage at 25°C, the ACE inhibitory activity retention rate remained at 85%, while in Comparative Example 4 (lacking tea polyphenol-zinc and Monascus purpureus extract), the activity retention rate decreased to 56%. The tea polyphenol-zinc chelate forms an antioxidant barrier through the coordination of phenolic hydroxyl groups with Zn²⁺, effectively inhibiting the oxidative degradation of peptides during storage. Simultaneously, MonacolinK (detected content ≥2.1 mg / g) in the Monascus purpureus extract synergistically acts with corn peptides at both ACE enzyme and HMG-CoA reductase dual targets, and this synergistic effect maintains the stability of the active conformation during long-term storage.

[0076] The synergistic effect of magnetic separation and stabilizer formulation further enhances the functional orientation of the final product. In Example 1, the recovered peptides naturally enrich ACE-inhibiting active sites due to affinity adsorption, while the charge properties of the stabilizer micelles (such as the electrostatic complexation of sodium alginate and L-arginine) not only improve physical stability but also enhance the affinity of peptides for intestinal transport proteins through zeta potential regulation. This end-to-end design, from separation to protection, overcomes the technical bottlenecks of easy inactivation of active ingredients and low oral bioavailability in traditional processes, providing an innovative solution for the development of functional foods.

[0077] Test Example 3: Comparative groups: Example 2, Comparative Example 5 (without Monascus purpureus), and Comparative Example 6 (eluted at pH 7.0).

[0078] Experimental steps: Sample preparation: Corn peptide compositions were prepared according to the methods described in Example 2, Comparative Example 5, and Comparative Example 6, with three batches prepared in parallel for each example.

[0079] Elution efficiency determination: Target peptide to total peptide mass ratio: Take the eluent after magnetic separation and the unadsorbed enzymatic hydrolysate (diluted to the same concentration), determine the total peptide content by BCA method, and calculate the target peptide ratio = (elution peptide mass / total peptide mass) × 100%.

[0080] Evaluation of the synergistic blood pressure lowering effect: ACE inhibition rate: determined according to the method in Test Example 1; HMG-CoA reductase inhibition rate: Take the sample solution (1 mg / mL) and react it with HMG-CoA reductase (0.1 U / mL) at 37℃ for 30 minutes, detect the rate of decrease in NADPH absorbance (340 nm), and calculate the inhibition rate; Synergistic effect value: ACE inhibition rate (%) × HMG-CoA inhibition rate (%) / 100.

[0081] Determination of zinc ion chelation rate: Atomic absorption spectrometry: Centrifuge the stabilizer-prepared solution (10,000×g, 10 minutes), filter the supernatant through a 0.45μm filter membrane, and determine the free Zn²⁺ concentration (wavelength 213.9nm). Chelation rate calculation: Chelation rate (%) = (Total Zn) 2+ -Free Zn 2+ ) / Total Zn 2+ ×100%.

[0082] Experimental data: Table 3. Effects of lower limit parameters, process parameters, and key components on the properties of corn peptides. Key points of data analysis: The criticality of acidic elution conditions: The elution efficiency of Example 2 (~75%) was significantly higher than that of Comparative Example 6 (~43%), indicating that the glycine-HCl buffer at pH 2.5 can effectively disrupt the hydrogen bond binding between the affinity ligand and the target peptide, while neutral conditions (pH 7.0) prevent the adsorbed peptide from being fully eluted.

[0083] Synergistic antihypertensive effect of Monascus purpureus extract: The synergistic effect value (~61%) of Example 2 was about 50% higher than that of Comparative Example 5 (~41%), demonstrating that MonacolinK in Monascus purpureus forms a multi-pathway antihypertensive mechanism by inhibiting HMG-CoA reductase and the ACE inhibitory activity of corn peptide.

[0084] Tea polyphenol-zinc chelation stability: chelation rates were all >85%, indicating that tea polyphenols and zinc chelate stability are good. 2+ The coordination effect is stable in the complex system, regardless of whether Monascus purpureus is added or the elution conditions.

[0085] Experimental Summary: The lower limit process of this invention achieves efficient target peptide recovery and multi-pathway depressant activity by optimizing biomimetic affinity separation and the synergistic effect of functional components. Experimental data show that Example 2, using acidic elution conditions at pH 2.5, achieved a significantly higher elution efficiency (~75%) than the neutral conditions (Comparative Example 6, ~43%). This is attributed to the reversible dissociation of hydrogen bonds and hydrophobic interactions between the Captopril analog and the target peptide in an acidic environment. The low pH of the glycine-HCl buffer protonates the carboxylic acid groups of the ligands, weakening their electrostatic attraction to basic amino acids (such as Arg and Lys) in the peptide, thereby releasing highly active peptides. In contrast, the neutral conditions (pH 7.0) cause the target peptides to remain on the surface of the magnetic microspheres due to excessively strong affinity.

[0086] The introduction of Monascus purpureus extract significantly enhanced the synergistic antihypertensive effect of corn peptides. The synergistic effect value (ACE × HMG-CoA inhibition rate, ~61%) in Example 2 was nearly 50% higher than that in Comparative Example 5 (without Monascus purpureus, ~41%), indicating that MonacolinK reduces cholesterol synthesis by inhibiting HMG-CoA reductase, forming a dual-pathway regulatory mechanism of "vasodilation-lipid metabolism" with the ACE-inhibiting activity of corn peptides. This synergistic effect relies on the conformational complementarity between MonacolinK (detected content ≥2.0 mg / g) in Monascus purpureus extract and the active site of corn peptides. Specifically, the six-membered lactone ring of MonacolinK enhances the hydrophobic binding of the peptide to the S2 subunit of ACE enzyme, while its side-chain hydroxyl groups form a hydrogen bond network with the NADPH-binding domain of HMG-CoA reductase.

[0087] The stabilizing coordination effect of the tea polyphenol-zinc chelate was fully verified in this process. The zinc ion chelation rates in Example 2 and Comparative Examples 5-6 were both >85%, indicating that the catechol structure of tea polyphenols forms a stable six-membered ring chelate through O-Zn coordination bonds, and its antioxidant activity is unaffected by elution conditions or the absence of Monascus purpureus components. This chelation not only inhibits the oxidative degradation of peptides but also enhances the intestinal absorption efficiency of bioactive peptides through zinc ion delivery, further strengthening the bioavailability of the final product. The above mechanistic correlation demonstrates that this invention achieves an integrated design of bioactive peptide separation, stabilization, and functional enhancement through precise matching of process parameters and functional components.

[0088] Test Example 4: Comparative groups: Example 3, Comparative Example 7 (without third-stage microwave), Comparative Example 8 (ultrafiltration replacing magnetic separation).

[0089] Experimental steps: Molecular weight distribution determination: HPLC-MS analysis: The purified corn peptide solution (1 mg / mL) was subjected to ultrafiltration centrifugation (3 kDa membrane) to remove macromolecular impurities. Separation was performed using a C18 column (2.1 × 150 mm, 1.7 μm) with mobile phase A (0.1% formic acid water) - B (0.1% formic acid acetonitrile) and gradient elution (5% B → 60% B, 30 min) at a flow rate of 0.3 mL / min. Mass spectrometry data (m / z 100-2000) were acquired in ESI positive ion mode, and the proportion of peptides <1kDa was statistically analyzed by molecular weight extraction ion chromatogram.

[0090] Stomach acid tolerance test: Simulated gastric juice processing: Take corn peptide composition (containing stabilizer) and simulated gastric juice (0.1MHCl, containing 3.2mg / mL pepsin) at a volume ratio of 1:1, and react at 37℃ with shaking (120rpm) for 2 hours; Add 0.1M NaHCO3 to neutralize to pH 6.8, centrifuge (10,000×g, 10 minutes) and collect the supernatant; ACE inhibitory activity retention rate: The ACE inhibition rate before and after treatment was determined according to the method in Test Example 1. The retention rate was calculated as (inhibition rate after treatment / initial inhibition rate) × 100%.

[0091] Transmembrane absorption rate determination: Caco-2 cell model: Caco-2 cells were seeded into Transwell plates (3 μm pores) and cultured for 21 days to form a complete monolayer (TEER value ≥ 500 Ω·cm). 2 ); Add corn peptide solution (0.5 mg / mL, HBSS buffer, pH 6.5) to the top side (AP chamber) and HBSS buffer (pH 7.4) to the bottom side (BL chamber). Incubate at 37°C for 2 hours, take the solution from the BL chamber, and determine the peptide concentration using the BCA method; Apparent permeability coefficient (Papp): Papp (×10) -6 cm / s) = (BL chamber peptide mass × monolayer area) / (AP chamber initial concentration × time).

[0092] Experimental data: Table 4. Effects of parameter upper limits and process substitutions on the performance of corn peptides Key points of data analysis: The promotion of hydrophobic peptide release by terminal microwaves: The proportion of <1kDa peptides in Example 3 (~90%) was significantly higher than that in Comparative Example 7 (~75%), demonstrating that the low-power microwaves (150W) in the third stage can disrupt the secondary bonds of the hydrophobic peptide-enzyme complex and promote the dissolution of small molecule active peptides.

[0093] Stabilizer charge-matched gastric acid protection mechanism: The gastric acid activity retention rate of Example 3 (~91%) was higher than that of Comparative Example 8 (~74%), indicating that sodium alginate-L-arginine micelles (Zeta potential +15~20mV) reduce the enzymatic attack of pepsin on peptides through electrostatic repulsion.

[0094] Optimization of transmembrane absorption by magnetic separation: Transmembrane absorption rate (Papp ~ 18 × 10⁻⁶) in Example 3 -6 cm / s) is the comparison example 8 (ultrafiltration, ~9×10) -6The magnetic separation efficiency (cm / s) is twice that of the magnetic separation efficiency (cm / s), indicating that the selectively enriched peptides (such as Leu-Pro-Pro) are more likely to bind to intestinal transport proteins (such as PepT1), while the ultrafiltration process may disrupt the active conformation.

[0095] Experimental Summary: This invention, through parameter upper limit process design and process substitution comparison, verifies the key role of dynamic temperature-assisted microwave release in the release of hydrophobic bioactive peptides. Experimental data show that in Example 3, after applying 150W pulsed microwaves in the final enzymatic hydrolysis stage (comparative Example 7 without microwaves), the proportion of <1kDa peptides increased to approximately 90%, significantly higher than the 75% in Comparative Example 7. This difference stems from the synergistic mechanism of the non-thermal effect of microwaves and the temperature gradient: the low-temperature (37℃) environment in the third stage allows the hydrogen bond anchoring of glutaminase to dominate, while the electromagnetic oscillation of low-power microwaves can directionally disrupt the π-π stacking of hydrophobic peptides and enzymatic hydrolysis byproducts, promoting the dissociation of small molecule bioactive peptides from the complex, while avoiding peptide chain oxidation and breakage caused by high temperatures.

[0096] The charge-matching mechanism of stabilizers plays a central role in gastric acid tolerance. The gastric acid activity retention rate of Example 3 (~91%) was approximately 17% higher than that of Comparative Example 8 (ultrafiltration process, ~74%), indicating that the sodium alginate-L-arginine complex micelles, through positive charge enrichment (Zeta potential +15~20mV), form electrostatic repulsion with the negatively charged gastric mucus layer, reducing the probability of pepsin approaching the peptides. In contrast, the ultrafiltration process, lacking biomimetic magnetic separation, exposes active peptides to mechanical shear forces during filtration, causing partial unwinding of the α-helix structure, making them more easily recognized and degraded by pepsin.

[0097] The bioavailability advantage of the magnetic separation process is confirmed by transmembrane uptake data. The Papp value of Example 3 is ~18 × 10⁻⁶. -6 cm / s) is the comparison example 8 (ultrafiltration, ~9×10) -6 The efficiency is twice that of hydrophobic peptides (cm / s), attributed to the fact that the hydrophobic peptides selectively enriched by affinity magnetic separation (such as Leu-Pro-Pro) have a complete tertiary conformation. Their amphiphilic structure can passively diffuse through the hydrophobic region of the cell membrane lipid bilayer and specifically bind to the hydrophobic binding domain of the intestinal transporter PepT1. In contrast, ultrafiltration processes, due to the lack of targeted separation, make it difficult for polar peptides mixed in the product to be effectively transported across the membrane. The above mechanism indicates that this invention, through process parameter optimization and function-oriented design, achieves a complete chain innovation of efficient release, stable protection, and targeted absorption of bioactive peptides.

[0098] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing corn peptides that lower blood pressure, characterized in that, Includes the following steps: S1. Raw material pretreatment: Corn protein is extracted by alkali dissolution and acid precipitation, and then subjected to microwave pretreatment; S2, Dynamic Temperature Synergistic Enzymatic Hydrolysis: The pretreated corn protein is subjected to three stages of enzymatic hydrolysis: low temperature, high temperature, and low temperature. Different proteases are added in each stage, and microwave assistance is applied at different times. S3. In-situ magnetic separation and purification: Surface-modified magnetic materials are added to the enzymatic hydrolysate to adsorb the target peptide fragments, followed by magnetic separation and elution. S4. Stabilizer compounding: The purified corn peptides are mixed with stabilizers and dried to obtain the final product.

2. The method for preparing a blood pressure-lowering corn peptide according to claim 1, characterized in that, The dynamic temperature-controlled synergistic enzymatic hydrolysis in step S2 specifically includes: First stage: At 43-47℃, add alkaline protease at an enzyme-to-protein ratio of 1:40-1:60 (w / w) and simultaneously apply pulsed microwaves; Second stage: Heat to 53-57℃, add chymotrypsin at an enzyme-to-protein ratio of 1:80-1:120 (w / w), and turn off the microwave. Third stage: Cool down to 33-37℃, add glutaminase, enzyme-to-protein ratio is 1:70-1:90 (w / w), and apply low-power microwave in the final stage.

3. The method for preparing a blood pressure-lowering corn peptide according to claim 1, characterized in that, In step S2, the microwave assistance must meet the following conditions: The microwave power in the first stage is 180-220W, the pulse period is 8-12 seconds, and the action time is ≤5 minutes. The third stage uses a microwave power of 100-150W, a pulse period of 5-8 seconds, and an action time of 8-12 minutes.

4. The method for preparing a blood pressure-lowering corn peptide according to claim 1, characterized in that, In step S3, the magnetic material is aminated Fe3O4@SiO2 microspheres with ACE inhibitory peptide affinity ligands grafted onto their surface. The affinity ligands are mercaptopropionylglycine or Captopril analogs.

5. The method for preparing a blood pressure-lowering corn peptide according to claim 1, characterized in that, The three-stage enzymatic hydrolysis times in step S2 are as follows: The first stage lasts 25-35 minutes, the second stage lasts 50-70 minutes, and the third stage lasts 25-35 minutes.

6. The method for preparing a blood pressure-lowering corn peptide according to claim 1, characterized in that, In step S3, the amount of magnetic material added is 4-6% (w / w) of the total mass of the enzymatic hydrolysate, and the elution buffer used in the elution process is a glycine-HCl solution with a pH of 2.5-3.

5.

7. The method for preparing a blood pressure-lowering corn peptide according to claim 1, characterized in that, The stabilizer in step S4 comprises the following components by mass parts: Sodium alginate 0.3-0.7 parts, L-arginine 0.7-1.3 parts, tea polyphenol-zinc chelate 0.1-0.3 parts, Monascus purpureus extract 0.05-0.15 parts.

8. The method for preparing a blood pressure-lowering corn peptide according to claim 7, characterized in that, The Monascus purpureus extract was prepared by the following steps: Mix the solid fermentation product of Monascus purpureus with 50-70% ethanol solution at a material-to-liquid ratio of 1:10-1:20 (w / v) and extract at 50-60℃ for 1-2 hours. After filtration, the extract is concentrated to 10-20% of its original volume to obtain an extract with a MonacolinK mass percentage of ≥0.2%.

9. The application of a blood pressure-lowering corn peptide prepared by the preparation method according to any one of claims 1-8 in the preparation of blood pressure-lowering functional foods, characterized in that, The functional foods mentioned are solid beverages, compressed candies, or dairy products.

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