A method for preparing a blood pressure lowering protein active peptide composition

Abstract

This application relates to the field of bioactive peptide preparation technology, and discloses a method for preparing a blood pressure-lowering protein active peptide composition. The raw materials for this composition include a protein substrate, deionized water, alkaline protease, a complex solubilizing buffer, and a cross-linked aqueous solution containing sodium hexametaphosphate. The preparation method includes: isothermal enzymatic hydrolysis of the protein substrate; adding the complex solubilizing buffer to the hydrolysate and instantaneously heating it to construct a steric hindrance protective layer for the short peptides; subsequently injecting the cross-linked aqueous solution for high-shear cross-linking, allowing large molecular impurities to target and bind; and then directly spraying it into a vacuum flash evaporator for rapid cooling, causing the cross-linked protein network to shrink and densify; finally, centrifugation, ultrafiltration, concentration, and drying are performed to obtain the finished product. This invention prevents the active short peptides from co-precipitating with impurities under heat, effectively maintaining the blood pressure-lowering activity of the peptides, and densifying the impurity flocs, thereby improving the separation and sludge removal efficiency and the anti-fouling ability of the ultrafiltration membrane.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide preparation technology, specifically a method for preparing a blood pressure-lowering protein bioactive peptide composition. Background Technology

[0002] Antihypertensive peptides are a class of functional short peptides that can inhibit the activity of angiotensin-converting enzyme. Because they are derived from natural proteins, they have the characteristics of high safety and easy absorption, and have high application value in the field of functional foods. At present, in industry, antihypertensive peptides are usually prepared by using proteins rich in relevant amino acid sequences as substrates and hydrolyzing them with specific proteases.

[0003] In traditional enzymatic extraction processes, to terminate the enzymatic reaction and kill microorganisms in the system, the feed solution is usually subjected to high-temperature enzyme inactivation after the target degree of hydrolysis is reached. Under high-temperature thermal shock, incompletely hydrolyzed macromolecular protein residues and various medium-molecular-weight peptides in the reaction system undergo thermal denaturation, disrupting the internal spatial conformational interactions and exposing a large number of hydrophobic amino acid residues. During this process, the target antihypertensive short peptides already released in the feed solution also undergo conformational changes in the heated environment, with their nonpolar regions also exposed in the aqueous phase. These exposed hydrophobic groups spontaneously generate hydrophobic interactions and electrostatic flocculation, causing highly active free short peptides to cross-bind and co-precipitate with macromolecular proteins. This non-specific aggregation causes the target short peptides to be encapsulated by impurities and discharged with the waste residue, reducing the peptide extraction yield. At the same time, the bioactivity of antihypertensive short peptides depends on the spatial chelation between their specific amino acid sequences and the active site of angiotensin-converting enzyme. Thermal aggregation and impurity adsorption mask the key binding sites of the short peptides, resulting in a decline in the antihypertensive activity of the final peptide product.

[0004] Furthermore, traditional processes face hydrodynamic separation challenges when handling such complex mixtures. The protein impurity flocs that spontaneously form upon heating have a loose and expanded structure, containing a large amount of bound water and short peptide molecules. In subsequent solid-liquid separation stages, these low-density flocs are difficult to completely settle using mechanical centrifugation, resulting in the overflow of tiny suspended particles and colloidal substances with the clarified liquid. When a feed solution containing a large number of protein particles enters the ultrafiltration membrane separation system, large molecular impurities migrate to the membrane interface under operating pressure and adsorb onto the membrane pores, forming a dense gel deposition layer on the membrane surface. This not only causes severe concentration polarization and irreversible membrane pore blockage, leading to a rapid decline in the permeate flux of the ultrafiltration system, but also increases the frequency of chemical cleaning of the membrane module and operational energy consumption. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing a blood pressure-lowering protein active peptide composition. This method solves the problems in traditional peptide preparation processes, such as the target short peptide easily undergoing hydrophobic cross-linking and co-precipitation with impurities during the high-temperature treatment stage, leading to reduced short peptide activity and yield. It also addresses the issues of loose impurity flocs causing incomplete solid-liquid separation, which in turn leads to irreversible fouling and rapid flux decline in subsequent ultrafiltration membranes.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a blood pressure-lowering protein active peptide composition having a locked hydrophobic short peptide spatial conformation to inhibit the activity of angiotensin-converting enzyme. The composition is made from raw materials comprising the following parts by weight: Protein substrate: 10-15 parts by weight; Deionized water: 100 parts by weight; Alkaline protease: 0.02-0.05 parts by weight; Compound solubilizing buffer: 5-10 parts by weight; Crosslinked aqueous solution: prepared from 10 parts by weight of deionized water and 0.15-0.25 parts by weight of sodium hexametaphosphate.

[0007] By adopting the above technical solution, and using a specific ratio of composite solubilizing buffer solution combined with sodium hexametaphosphate crosslinked aqueous solution, the reaction mechanism is as follows: Sodium hexametaphosphate, as a polyanionic crosslinking agent, specifically targets macromolecular protein remnants within the system at this formulation. Under extreme high-temperature conditions, the macromolecular proteins undergo thermal denaturation and conformational unwinding, exposing their internal positively charged regions. Sodium hexametaphosphate then binds to the macromolecular proteins through electrostatic bridging.

[0008] The antihypertensive effect depends on the spatial chelation between the hydrophobic amino acid residues at the C-terminus of the short peptide and the zinc ion at the active site of angiotensin-converting enzyme. The synergistic effect of these components avoids the non-specific aggregation of the peptide fragment under thermal conditions, thus maintaining the natural compact structure of the hydrolysis product. The short peptide sequence is protected from oxidative damage and physical masking, and its half-maximal inhibitory concentration (IC50) against angiotensin-converting enzyme remains at a low level.

[0009] Preferably, the solute in the composite solubilizing buffer is composed of L-arginine and anhydrous citric acid; the molar ratio of L-arginine to anhydrous citric acid in the composite solubilizing buffer is 2.2-2.8:1; and the concentration of L-arginine in the composite solubilizing buffer is 1.0 mol / L.

[0010] By employing the above technical solution, L-arginine carries a highly polar guanidine group in its molecular structure, while citric acid provides a multi-carboxyl group structure. When the two are mixed in a molar ratio of 2.2-2.8:1, they form a salt complex in the aqueous phase. The guanidine group of L-arginine associates with water molecules through hydrogen bonding, constructing a hydration layer on the surface of the hydrophobic short peptide. The carboxyl anion of citric acid stabilizes this hydration layer structure through electrostatic repulsion. The specific molar ratio ensures the presence of an appropriate amount of free protons and polar groups in the system, avoiding osmotic pressure imbalance caused by excessive solubilizer. The 1.0 mol / L L-arginine concentration provides sufficient molecular collision probability in the enzymatic hydrolysis solution, allowing the solubilizing buffer molecules to quickly cover the surface of the target short peptide and enhance the freeness of the short peptide in a high-temperature environment.

[0011] Preferably, the protein substrate is selected from casein or soy protein isolate; the initial addition temperature of the deionized water is 48-52°C; and the cross-linked aqueous solution is prepared by mixing and dissolving the deionized water with the sodium hexametaphosphate.

[0012] By adopting the above technical solution, casein and soy protein isolate are used as precursor materials containing characteristic antihypertensive hydrophobic amino acid fragments with corresponding sequences. The initial water temperature of 48-52℃ puts the protein substrate in a swollen state, increasing the accessibility of internal enzyme cleavage sites. Sodium hexametaphosphate is pre-dissolved in deionized water to form a uniform liquid crosslinking agent, avoiding the phenomenon of excessively high local concentration and uneven crosslinking caused by directly adding solid powder to the liquid. This ensures that polyphosphate molecules can diffuse uniformly and synchronously electrostatically bridge with macromolecular protein residues.

[0013] Preferably, the alkaline protease is an alkaline protease extracted by fermentation of Bacillus subtilis or an alkaline protease extracted by fermentation of Bacillus licheniformis; the enzyme activity of the alkaline protease is 200,000-400,000 U / g.

[0014] By employing the above-mentioned technical solution, alkaline proteases from specific microbial sources exhibit specific endonuclease activity on peptide bonds in substrates, tending to cleave at the side chains of hydrophobic amino acid residues, thus promoting the release of short, blood pressure-lowering peptides. The enzyme activity range of 200,000-400,000 U / g ensures sufficient catalytic active sites in the reaction system, maintaining high substrate conversion efficiency with a trace addition of 0.02-0.05 parts by weight, controlling the degree of polypeptide chain cleavage, avoiding excessive hydrolysis that could lead to sequence breakage, and simultaneously reducing the amount of exogenous protein introduced.

[0015] This invention provides a method for preparing a blood pressure-lowering protein active peptide composition, comprising the following steps: The protein substrate was added to preheated deionized water, and after adjusting the pH of the system, alkaline protease was added for constant temperature and dynamic pH adjustment of the stirring enzymatic hydrolysis. After the target degree of hydrolysis was reached, the enzymatic hydrolysate was obtained. The composite solubilizing buffer is added to the enzymatic hydrolysis solution, and after being stirred and mixed at a constant temperature, it is continuously fed into a heat exchanger for instantaneous heating to obtain a high-temperature solution. The crosslinking aqueous solution is injected into a high-shear mixing device located at the outlet of the heat exchanger. After instantaneous crosslinking with the high-temperature liquid, it is directly sprayed into a vacuum flash evaporation device to cause the mixed system to boil and vaporize and cool down instantly. After staying at the bottom of the tank, the modified crosslinking liquid is obtained. The denatured cross-linked liquid was subjected to mechanical solid-liquid separation, ultrafiltration cross-flow impurity removal, evaporation concentration and spray drying in sequence to obtain the final blood pressure lowering protein active peptide composition.

[0016] Preferably, the complex solubilizing buffer is prepared in advance through the following steps: Add the prescribed amount of anhydrous citric acid to deionized water and stir mechanically at room temperature until completely dissolved to obtain an aqueous citric acid solution. Under continuous stirring, the prescribed amount of L-arginine was added to the citric acid aqueous solution, and stirring was continued until the solids were completely dissolved. Add deionized water to make up the volume.

[0017] By adopting the above technical solution, anhydrous citric acid and L-arginine are dissolved sequentially at room temperature. The exothermic reaction of acid-base neutralization and the kinetic energy of mechanical stirring promote the hydrolysis and dissociation of solute molecules. The room temperature operation avoids the deamination and degradation of L-arginine under heating conditions, ensuring the structural integrity of the polar functional groups in the buffer solution, so that it can stably play the role of electrostatic shielding and solubilizing when enzymatic hydrolysis solution is added later.

[0018] Preferably, the protein substrate is added to preheated deionized water, the pH of the system is adjusted, and then alkaline protease is added for enzymatic hydrolysis with constant temperature and dynamic pH adjustment. The enzymatic hydrolysis solution is obtained after reaching the target degree of hydrolysis. Specifically, the process includes: adjusting the initial pH of the deionized water and protein substrate mixture to 8.0 using sodium hydroxide aqueous solution; adding alkaline protease to the mixture and performing enzymatic hydrolysis with stirring under constant temperature conditions; dynamically adding sodium hydroxide aqueous solution during the enzymatic hydrolysis process to maintain the pH of the system at 8.0; and stopping the addition of sodium hydroxide aqueous solution when the degree of hydrolysis of the system reaches 15-18.

[0019] Preferably, the system with added compound solubilizing buffer is stirred for 15-20 minutes at a temperature of 48-52℃ and a speed of 50-100 rpm; the stirred material is continuously pumped into a shell-and-tube heat exchanger; the liquid is controlled to stay in the tube side of the shell-and-tube heat exchanger for 15-20 seconds, so that the temperature of the liquid at the outlet of the shell-and-tube heat exchanger reaches 93-97℃.

[0020] By adopting the above technical solution, the mild conditions of 48-52℃ combined with medium and low speed stirring provide sufficient mass transfer time for the diffusion and penetration of buffer molecules to the surface of the target short peptide, completing the initial construction of the steric hindrance layer. The 15-20 second short-time high-temperature treatment provided by the tube heat exchanger not only meets the activation energy required to inactivate residual alkaline proteases and promote the thermal denaturation and unfolding of macromolecular proteins, but also compresses the heat exposure time to an extremely short range, blocking the thermal degradation pathway of the short peptide sequence under continuous high temperature.

[0021] Preferably, a pipeline-type static high-shear mixer is used as the high-shear mixing device, the shear rate of mixing and crosslinking is controlled at 3000-5000 s⁻¹, and the mixing and crosslinking time is 1.0-2.0 seconds; the absolute pressure of the vacuum flash evaporator is controlled to be constant at 0.02-0.03 MPa; the mixing system is cooled to 58-62°C within 1-3 seconds and remains at the bottom of the vacuum flash evaporator for 3-5 minutes.

[0022] By adopting the above technical solution, a strong shear rate of 3000-5000 s⁻¹ generates high-frequency turbulence in a very small space, and an extremely short cross-linking time of 1.0-2.0 seconds confines the reaction to the microscopic bridging stage, preventing the cross-linked material from growing excessively and forming gel blocks that clog the channels. The constant negative pressure of 0.02-0.03 MPa directly corresponds to the boiling point range of water at 58-62℃. Under this pressure, the flash vaporization of the material forcibly strips away the sensible heat contained in the system, triggering a rapid cooling of 1-3 seconds. The bottom residence process provides sufficient time for the cross-linked precipitate to rearrange its structure and shrink in density, thus completing the final solidification of the phase change reaction.

[0023] Preferably, the modified crosslinked feed liquid is continuously fed into a horizontal screw discharge centrifuge for mechanical solid-liquid separation at a separation factor of 3000-5000g, and the supernatant is collected. The supernatant is pumped into an ultrafiltration membrane module with a molecular weight cutoff of 3000 Da for ultrafiltration cross-flow impurity removal, and the permeate is collected. The permeate is fed into a mechanical vapor recompression evaporator for evaporation and concentration, and the concentration is controlled to contain 20-30 parts by weight of solids per 100 parts by weight of concentrate. The concentrated material is then spray-dried.

[0024] By adopting the above technical solution, the large cross-linked flocs after phase change densification are rapidly discharged under a centrifugal force field of 3000-5000g. The colloidal substances that cause membrane fouling have been basically eliminated in the centrifugal supernatant, ensuring that the ultrafiltration process with a molecular weight cutoff of 3000Da can maintain a high permeation flux. The residual small molecule impurities and target peptides are separated at the molecular level. Mechanical vapor recompression technology is used for evaporation and dehydration, which reduces the heat load of heat-sensitive peptides in the concentration stage. Finally, spray drying technology is used to transform the liquid phase composition into a physicochemically stable powder product.

[0025] This invention provides a method for preparing a blood pressure-lowering protein active peptide composition. It has the following beneficial effects: 1. This invention provides steric hindrance protection for the antihypertensive short peptides in the extraction system by adding a composite solubilizing buffer composed of L-arginine and anhydrous citric acid. During the heating treatment stage, the buffer molecules can coat the surface of the short peptides to form a hydration layer, preventing irreversible hydrophobic cross-linking caused by the exposure of nonpolar residues due to heat. This treatment method prevents the target highly active short peptides from co-precipitating with macromolecular impurities, maintains the compact structure of the short peptides and the antihypertensive binding sites, and improves the overall yield of the final peptide product.

[0026] 2. This invention introduces sodium hexametaphosphate under high-temperature unfolding conditions and combines it with high-shear mixing to achieve targeted cross-linking and precipitation of large molecular weight impurity proteins. In the high-speed flow field of the inline mixer, sodium hexametaphosphate rapidly binds to the positively charged regions inside the thermally denatured large molecular weight proteins to form a cross-linking network, while the free short peptides protected by the initial buffer are dispersed outside the cross-linking network with the fluid. This instantaneous flow field cross-linking mechanism avoids the target short peptides being physically encapsulated by the impurity protein gel, ensuring that the active short peptides can be fully released in the clear liquid phase.

[0027] 3. This invention utilizes vacuum flash evaporation technology to combine thermodynamic phase change with flocculation and densification processes, improving the hydrodynamic filtration performance of the feed solution in the separation and purification process. The instantaneous boiling generated when the high-temperature mixed feed solution is injected into a negative pressure environment forces the liquid phase to cool and shrink rapidly. The resulting physical stress forcibly compresses the originally loose cross-linked network of proteins into a high-density solid precipitate, squeezing out the free water and target peptides entrained within. This densification process improves the separation efficiency of downstream mechanical centrifugation, removes tiny suspended particles that easily cause membrane fouling, alleviates the clogging problem of ultrafiltration membrane pores, and maintains the permeate flux of the membrane separation system during long-term operation. Attached Figure Description

[0028] Figure 1 This is a graph showing the relationship between the aggregation state and water content of the system in this invention. Figure 2 This is a comparison diagram of the residual peak areas of volatile substances in this invention; Figure 3 This is a comparison chart of the total yield and the distribution ratio of short peptides in this invention; Figure 4 This is a comparison chart of the blood pressure-lowering bioactivity indicators of the present invention; Figure 5 This is a comparison chart of the separation efficiency of the ultrafiltration membrane of the present invention. Detailed Implementation

[0029] 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.

[0030] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0031] Casein, CAS No. 9000-71-9, food grade, with a protein dry basis mass fraction of not less than 90.0%, a moisture mass fraction of not more than 8.0%, and an ash mass fraction of not more than 1.5%.

[0032] Soy protein isolate, CAS No. 9010-10-0, food grade, with a protein dry basis mass fraction of not less than 90.0% and a moisture mass fraction of not more than 7.0%.

[0033] Alkaline protease, derived from Bacillus subtilis, CAS No. 9014-01-1, with a nominal enzyme activity of 100,000 U / g to 200,000 U / g.

[0034] L-arginine, CAS No. 74-79-3, purity not less than 99.0%.

[0035] Anhydrous citric acid, CAS No. 77-92-9, purity not less than 99.5%.

[0036] Preparation Examples 1-3: Preparation Example 1: This preparation example provides a method for preparing a composite solubilizing buffer, including the following steps: Accurately weigh 2.5 mol of L-arginine and 1.0 mol of anhydrous citric acid. At this point, the molar ratio of L-arginine to anhydrous citric acid is 2.5:1. Measure an appropriate amount of deionized water and place it in a mixing tank. Add the weighed anhydrous citric acid to the deionized water and turn on the mechanical stirring at room temperature until it is completely dissolved to obtain a citric acid aqueous solution. Under continuous stirring, the weighed L-arginine was slowly added to the citric acid aqueous solution, and stirring was continued until the solid was completely clear and dissolved. Finally, the volume was adjusted to 2500 mL with deionized water to obtain a composite solubilizing buffer with an L-arginine concentration of 1.0 mol / L for later use.

[0037] Preparation Example 2: This preparation example provides a method for preparing a composite solubilizing buffer, including the following steps: Accurately weigh 2.2 mol of L-arginine and 1.0 mol of anhydrous citric acid. At this point, the molar ratio of L-arginine to anhydrous citric acid is 2.2:1. Measure an appropriate amount of deionized water and place it in a mixing tank. Add the weighed anhydrous citric acid to the deionized water and turn on the mechanical stirring at room temperature until it is completely dissolved to obtain a citric acid aqueous solution. Under continuous stirring, the weighed L-arginine was slowly added to the citric acid aqueous solution, and stirring was continued until the solid was completely clear and dissolved. Finally, the volume was adjusted to 2200 mL with deionized water to obtain a composite solubilizing buffer with an L-arginine concentration of 1.0 mol / L for later use.

[0038] Preparation Example 3: This preparation example provides a method for preparing a composite solubilizing buffer, including the following steps: Accurately weigh 2.8 mol of L-arginine and 1.0 mol of anhydrous citric acid. At this point, the molar ratio of L-arginine to anhydrous citric acid is 2.8:1. Measure an appropriate amount of deionized water and place it in a mixing tank. Add the weighed anhydrous citric acid to the deionized water and turn on the mechanical stirring at room temperature until it is completely dissolved to obtain a citric acid aqueous solution. Under continuous stirring, the weighed L-arginine was slowly added to the citric acid aqueous solution, and stirring was continued until the solid was completely clear and dissolved. Finally, the volume was adjusted to 2800 mL with deionized water to obtain a composite solubilizing buffer with an L-arginine concentration of 1.0 mol / L for later use.

[0039] Examples 1-4: Example 1: This embodiment provides a method for preparing a blood pressure-lowering protein active peptide composition, comprising the following steps: Add 12 parts by weight of casein to 100 parts by weight of deionized water preheated to 50°C, adjust the pH to 8.0 with sodium hydroxide aqueous solution, add 0.04 parts by weight of alkaline protease, and stir to hydrolyze until the degree of hydrolysis reaches 16.5 under constant temperature and dynamic replenishment of sodium hydroxide aqueous solution to maintain the pH at 8.0. Stop adding alkaline solution.

[0040] Eight parts by weight of the composite solubilizing buffer prepared in Preparation Example 1 were pumped into the above enzymatic hydrolysis solution. The solution was stirred at 50 rpm for 15 minutes at 50°C. The solution was then continuously pumped into a shell-and-tube heat exchanger so that it remained in the tubes for 15 seconds and reached 95°C at the outlet.

[0041] An aqueous solution prepared from 10 parts by weight of deionized water and 0.2 parts by weight of sodium hexametaphosphate is injected into a pipeline static high-shear mixer located at the outlet of a shell-and-tube heat exchanger. It is instantaneously mixed and crosslinked with the high-temperature liquid at a shear rate of 4000 s⁻¹ for 1.5 seconds. The mixture is then directly sprayed into a vacuum flash tank with an absolute pressure of 0.025 MPa, causing the mixture to boil instantly and cool down to 60°C within 2 seconds, where it remains at the bottom of the vacuum flash tank for 4 minutes.

[0042] The liquid material at the bottom of the vacuum flash tank is continuously fed into a horizontal screw discharge centrifuge for solid-liquid separation at a separation factor of 4000g. The supernatant is collected and pumped into an ultrafiltration membrane module with a molecular weight cutoff of 3000 Da for cross-flow filtration. The permeate is collected and fed into a mechanical vapor recompression evaporator for concentration until 25 parts by weight of solids are contained in every 100 parts by weight of the concentrate. After spray drying, a blood pressure lowering protein active peptide composition is obtained.

[0043] Example 2: This embodiment provides a method for preparing a blood pressure-lowering protein active peptide composition, comprising the following steps: Add 10 parts by weight of casein to 100 parts by weight of deionized water preheated to 48°C, adjust the pH to 8.0 with sodium hydroxide aqueous solution, add 0.02 parts by weight of alkaline protease, and stir to hydrolyze until the degree of hydrolysis reaches 15 under constant temperature and dynamic replenishment of sodium hydroxide aqueous solution to maintain the pH at 8.0, then stop adding alkaline solution.

[0044] Five parts by weight of the composite solubilizing buffer prepared in Preparation Example 2 were pumped into the above enzymatic hydrolysis solution. The solution was stirred at 50 rpm for 15 minutes at 48°C. The solution was then continuously pumped into a shell-and-tube heat exchanger so that it remained in the tubes for 15 seconds and reached 93°C at the outlet.

[0045] An aqueous solution prepared from 10 parts by weight of deionized water and 0.15 parts by weight of sodium hexametaphosphate is injected into a pipeline static high-shear mixer located at the outlet of a shell-and-tube heat exchanger. It is instantaneously mixed and crosslinked with the high-temperature liquid at a shear rate of 3000 s⁻¹ for 1.0 second. The mixture is then directly sprayed into a vacuum flash tank with an absolute pressure of 0.03 MPa, causing the mixture to boil instantly and cool down to 62°C within 1 second, where it remains at the bottom of the vacuum flash tank for 3 minutes.

[0046] The liquid material at the bottom of the vacuum flash tank is continuously fed into a horizontal screw discharge centrifuge for solid-liquid separation at a separation factor of 3000g. The supernatant is collected and pumped into an ultrafiltration membrane module with a molecular weight cutoff of 3000 Da for cross-flow filtration. The permeate is collected and fed into a mechanical vapor recompression evaporator for concentration until 20 parts by weight of solids are contained in every 100 parts by weight of the concentrate. After spray drying, a blood pressure lowering protein active peptide composition is obtained.

[0047] Example 3: This embodiment provides a method for preparing a blood pressure-lowering protein active peptide composition, comprising the following steps: Add 15 parts by weight of casein to 100 parts by weight of deionized water preheated to 52°C, adjust the pH to 8.0 with sodium hydroxide aqueous solution, add 0.05 parts by weight of alkaline protease, and stir to hydrolyze until the degree of hydrolysis reaches 18 under constant temperature and dynamic replenishment of sodium hydroxide aqueous solution to maintain the pH at 8.0, then stop adding alkaline solution.

[0048] Ten parts by weight of the composite solubilizing buffer prepared in Preparation Example 3 were pumped into the above enzymatic hydrolysis solution. The solution was stirred at 100 rpm for 20 minutes at 52°C. The solution was then continuously pumped into a shell-and-tube heat exchanger so that it remained in the tubes for 20 seconds and reached 97°C at the outlet.

[0049] An aqueous solution prepared from 10 parts by weight of deionized water and 0.25 parts by weight of sodium hexametaphosphate is injected into a pipeline static high-shear mixer located at the outlet of a shell-and-tube heat exchanger. It is instantaneously mixed and crosslinked with the high-temperature liquid at a shear rate of 5000 s⁻¹ for 2.0 seconds. The mixture is then directly sprayed into a vacuum flash tank with an absolute pressure of 0.02 MPa, causing the mixture to boil instantly and cool down to 58°C within 3 seconds, where it remains at the bottom of the vacuum flash tank for 5 minutes.

[0050] The liquid material at the bottom of the vacuum flash tank is continuously fed into a horizontal screw discharge centrifuge for solid-liquid separation at a separation factor of 5000g. The supernatant is collected and pumped into an ultrafiltration membrane module with a molecular weight cutoff of 3000 Da for cross-flow filtration. The permeate is collected and fed into a mechanical vapor recompression evaporator for concentration until 30 parts by weight of solids are contained in every 100 parts by weight of the concentrate. After spray drying, a blood pressure lowering protein active peptide composition is obtained.

[0051] Example 4: This embodiment provides a method for preparing a blood pressure-lowering protein active peptide composition, comprising the following steps: Add 12 parts by weight of soy protein isolate to 100 parts by weight of deionized water preheated to 50°C, adjust the pH to 8.0 with sodium hydroxide aqueous solution, add 0.04 parts by weight of alkaline protease, and stir to hydrolyze until the degree of hydrolysis reaches 16.5 under constant temperature and dynamic replenishment of sodium hydroxide aqueous solution to maintain the pH at 8.0. Stop adding alkaline solution.

[0052] Eight parts by weight of the composite solubilizing buffer prepared in Preparation Example 1 were pumped into the above enzymatic hydrolysis solution. The solution was stirred at 50 rpm for 15 minutes at 50°C. The solution was then continuously pumped into a shell-and-tube heat exchanger so that it remained in the tubes for 15 seconds and reached 95°C at the outlet.

[0053] An aqueous solution prepared from 10 parts by weight of deionized water and 0.2 parts by weight of sodium hexametaphosphate is injected into a pipeline static high-shear mixer located at the outlet of a shell-and-tube heat exchanger. It is instantaneously mixed and crosslinked with the high-temperature liquid at a shear rate of 4000 s⁻¹ for 1.5 seconds. The mixture is then directly sprayed into a vacuum flash tank with an absolute pressure of 0.025 MPa, causing the mixture to boil instantly and cool down to 60°C within 2 seconds, where it remains at the bottom of the vacuum flash tank for 4 minutes.

[0054] The liquid material at the bottom of the vacuum flash tank is continuously fed into a horizontal screw discharge centrifuge for solid-liquid separation at a separation factor of 4000g. The supernatant is collected and pumped into an ultrafiltration membrane module with a molecular weight cutoff of 3000 Da for cross-flow filtration. The permeate is collected and fed into a mechanical vapor recompression evaporator for concentration until 25 parts by weight of solids are contained in every 100 parts by weight of the concentrate. After spray drying, a blood pressure lowering protein active peptide composition is obtained.

[0055] Comparative Examples 1-4: Comparative Example 1: Compared with Example 1, the difference is that after stopping the addition of alkali solution, the composite solubilizing buffer is not pumped in, nor is it fed into the shell-and-tube heat exchanger and vacuum flash tank for processing. Instead, the solution is directly heated to 95°C in the reactor and kept at that temperature for 15 minutes to inactivate the enzyme. Then it is naturally cooled to 60°C. Sodium hexametaphosphate aqueous solution is not added throughout the process. All other aspects are the same.

[0056] Comparative Example 2: Compared with Example 1, the difference is that after stopping the addition of alkali solution, the composite solubilizing buffer is not pumped in, but the enzymatic hydrolysate is directly pumped into the shell and tube heat exchanger. All other aspects are the same.

[0057] Comparative Example 3: Compared with Example 1, the difference is that after stopping the addition of alkali solution, the composite solubilizing buffer and sodium hexametaphosphate aqueous solution are added to the enzymatic hydrolysis solution in the reactor at the same time. The mixture is stirred at 50 rpm for 15 minutes at 50°C. Then, it is pumped into the shell and tube heat exchanger to make the solution reach 95°C and then directly sprayed into the vacuum flash tank. The high shear injection operation at the outlet of the shell and tube heat exchanger is not performed. All other aspects are the same.

[0058] Comparative Example 4: Compared with Example 1, the difference is that after the liquid is instantaneously mixed and crosslinked in the pipeline static high-shear mixer, it is not sprayed into the vacuum flash tank, but directly fed into the tubular cooler at atmospheric pressure, where it is cooled to 60°C by circulating cooling water and held for 4 minutes. All other aspects are the same.

[0059] Test Examples 1-5: Test Example 1: Test objective: To verify the effectiveness of the composite solubilizing buffer in the process of this application for locking the spatial conformation of hydrophobic short peptides, and the physical effect of the vacuum flash phase change quenching process on the macroscopic hydrophobic shrinkage and water-displacing ability of solid-phase crosslinking precipitation.

[0060] Experimental steps: For each embodiment and comparative example, after the system has completed the heat treatment and crosslinking process and before entering the mechanical centrifugation separation, a 15 mL liquid sample is taken, diluted with deionized water to the concentration required for instrument testing, and injected into the sample cell of the dynamic light scattering instrument.

[0061] The test temperature of the dynamic light scattering instrument was kept constant at 25℃, and the instrument was allowed to stand still for 120 seconds to eliminate internal convection interference before the scattering signal was collected.

[0062] The solid precipitate residues discharged from each group are continuously collected at the discharge port of the horizontal screw centrifuge. Approximately 5.0g of residue is spread evenly in an aluminum weighing dish of known mass and its initial wet weight is accurately measured.

[0063] Place the weighing dish containing the residue sample into an electric thermostatic drying oven set at 105℃ and bake until the difference between two consecutive weighings is less than the specified range. Then transfer it to a desiccator to cool to room temperature and weigh the final dry weight. Calculate the moisture content of the precipitated residue based on the mass loss.

[0064] The experimental results are shown in Table 1: Table 1: Test results of aggregate state and sediment water content in each example and comparative example ; in conclusion: Figure 1 This is a graph showing the relationship between the aggregation state and water content of the various embodiments and comparative systems of the present invention. Figure 1 The left-hand coordinate axis and the solid line marked with circles correspond to the Z-average particle size values ​​obtained from the liquid phase material tests in Examples 1-4 and Comparative Examples 1-4, while the right-hand coordinate axis and the dashed line marked with squares correspond to the moisture content values ​​of the solid precipitate residue after centrifugation in each corresponding group.

[0065] According to the data in Table 1, the Z-average particle size of the liquid phase materials in Examples 1 to 4 remained stable within a narrow range of 135-152 nm, while the polydispersity index of the system did not exceed 0.25, and the water content of the precipitate residue was suppressed to between 41% and 47%. Compared with Comparative Example 1, which used traditional enzyme inactivation and physical separation, the particle size of the system surged to a critical micron level approaching 1000 nm, and the centrifuged residue contained as much as 78.3% water, like a sponge.

[0066] This significant difference in physical morphology actually reflects the irreversible thermal collapse copolymerization phenomenon that occurs when hydrophobic short peptides cross the electrostatic repulsion barrier upon heating. Even with the introduction of a tubular transient enzyme inactivation system in Comparative Example 2, which can shorten the heating period, the lack of a protective composite buffer layer formed by L-arginine and citric acid in the liquid phase of the system means that short peptide residues still aggregate with large molecular debris, resulting in a particle size exceeding 800 nm.

[0067] Turning our attention to the control groups where the timing of reagent addition and process rhythm were disrupted, in Comparative Example 3, the room-temperature cross-linking under insufficient macromolecular defolding conditions not only resulted in a loose floc structure but also failed to effectively intercept microscopic peptide chains. Of particular note is Comparative Example 4, which retained all chemical systems and completed high-temperature, high-shear cross-linking but eliminated flash quenching. While the liquid phase particle size in this group was indeed maintained at the ideal level of 145.8 nm under chemical protection, the sludge moisture content rebounded to 65.4%.

[0068] This anomalous phenomenon suggests that conventional heat transfer and cooling methods are ineffective in reshaping the microscopic phase structure. Only the intense latent heat absorption process caused by negative pressure flash evaporation can alter the solvent dielectric constant on a millisecond scale. The rapid cooling and contraction effect resulting from this phase transition forces the polymer cross-linked materials to undergo intense physical densification. This process not only squeezes out bound water from the structure but also prevents highly active short peptides from being indiscriminately trapped and lost by loose flocs. Various chemical elements form a closed-loop support system and play a synergistic role in this process.

[0069] Test Example 2: Test objective: To verify the effect of vacuum phase change process on the in-situ simultaneous removal of characteristic bitter and fishy volatile organic compounds from the product when the latent heat of vaporization is rapidly cooled.

[0070] Experimental steps: Accurately weigh 2.0g of each of the blood pressure-lowering protein active peptide composition dry powders finally prepared in each example and comparative example, place them in a headspace sample bottle with a volume of 20mL, add 5.0mL of deionized water and vortex to completely redissolve them, add 2.0g of sodium chloride to reduce the solubility of volatile components in the aqueous phase and promote gas phase partitioning, and seal with an aluminum cap with a polytetrafluoroethylene septum.

[0071] The sample vial was placed in the constant temperature heating module and allowed to stand at 60°C for 20 minutes to reach equilibrium. The solid phase microextraction handle, which had undergone high temperature aging treatment, was then inserted through the septum and the fiber-coated head was pushed out to expose the air layer at the top of the sample vial. Adsorption was continued for 40 minutes under constant temperature conditions.

[0072] After adsorption was complete, the fiber head was retracted and the extraction handle was quickly transferred to the injection port of the gas chromatography-mass spectrometry (GC-MS) instrument. Thermal desorption was performed at an injection port temperature of 250°C for 5 minutes. Separation was performed using a weakly polar capillary column in the gas chromatography. The initial temperature of the column oven was set to 40°C and maintained for 3 minutes. The temperature was then programmed to increase to 150°C at a rate of 5°C per minute, followed by a rate of 10°C per minute to 240°C and a hold for 5 minutes.

[0073] The mass spectrometer detection end was set with an electron impact ion source energy of 70 eV and a scan mass range between mass-to-charge ratios of 35 and 350. Characteristic ion chromatograms corresponding to hexanal and 1-octen-3-ol were extracted. The baseline integration of the chromatographic peaks corresponding to the target substances was performed using the instrument’s built-in data processing workstation, and the corresponding absolute values ​​of the residual peak areas were recorded.

[0074] The experimental results are shown in Table 2: Table 2: Results of residual peak area tests of characteristic bitter and fishy odor volatile compounds in each example and comparative example. ; in conclusion: Figure 2 This is a comparison chart of the residual peak areas of volatile substances in various embodiments and comparative examples of the present invention. Figure 2 The horizontal axis corresponds to different groups, namely Examples 1 to 4 and Comparative Examples 1 to 4. The vertical axis represents the peak area values ​​obtained by gas chromatography-mass spectrometry integration. The solid lines marked with circles in the figure represent the residual peak area distribution of hexanal, and the dashed lines marked with squares represent the residual peak area distribution of 1-octen-3-ol.

[0075] According to the data in Table 2, the residual peak areas of hexanal and 1-octen-3-ol in Examples 1 to 4 are all in the extremely low range. During industrial-scale proteolytic hydrolysis, excessive oxidation of lipid residues within the substrate is often present. The large amount of highly hydrophobic small-molecule aldehydes and alcohols released by these reactions constitutes the source of the unbearable bitter and fishy odor in peptide formulations. These odor molecules not only have extremely low olfactory thresholds, but also tend to adhere to the exposed hydrophobic amino acid residues on the surface of antihypertensive active peptides through van der Waals forces and hydrophobic interactions. Conventional heating or crude separation methods are unlikely to remove them from the peptide binding sites.

[0076] Observing the data of Comparative Example 4, it is not difficult to find that after the key step of vacuum flash evaporation was omitted, although the liquid system underwent pipeline cooling flow, the chromatographic peak area of ​​hexanal increased sharply to 168453, and the peak area of ​​1-octen-3-ol also reached an astonishing 125632. This contrast clearly confirms that the instantaneous boiling and vaporization of materials under negative pressure provides far more than just simple heat conduction. When a liquid material at a temperature as high as 95°C is injected into a vacuum system maintained at extremely low absolute pressure, countless tiny vapor bubbles are formed inside the fluid within milliseconds. This intense gas-liquid phase mass transfer driving force is like a microscopic airflow scouring, directly breaking the non-covalent bond between volatile molecules and short peptides.

[0077] The bitter and fishy components that were forcibly desorbed were completely removed from the reaction system along with water vapor by the vacuum pump. The atmospheric pressure steady-state enzyme inactivation and cooling process used in Comparative Example 1 and Comparative Example 2 obviously lacked this instantaneous physical stripping energy, which resulted in this part of the odor substance being completely sealed in the subsequent solid-liquid separation liquid.

[0078] By introducing instantaneous decompression during the thermodynamic phase transition of the system, this method eliminates the need for the activated carbon deodorization step frequently used in subsequent processing. In routine peptide production, the addition of activated carbon can easily trigger non-specific adsorption of the target product, resulting in severe loss of activity. The phase transition quenching process avoids interference from exogenous adsorption materials, ensuring physical densification of the formulation while simultaneously improving the sensory purity of the final reconstituted dry powder.

[0079] Test Example 3: Test objective: To evaluate the effects of various chemical interventions and physical field coupling in the preparation process on the directional retention capacity of highly active antihypertensive hydrophobic short peptides and the overall peptide yield.

[0080] Experimental steps: Accurately weigh 100 mg of each of the blood pressure-lowering protein active peptide composition dry powder collected in each example and comparative example, place them in a 10 mL volumetric flask, add deionized water to dissolve and make up to volume, and sonicate for 5 minutes at room temperature to ensure that the solids inside the dry powder are completely released into the aqueous phase.

[0081] Using a syringe, draw up the above reconstituted solution and slowly push it through a polyethersulfone microporous filter membrane with a pore size of 0.22 μm. After discarding the first two drops of initial filtrate, collect the subsequent clear liquid into a sample vial with a Teflon-lined cap and wait for instrument testing.

[0082] Molecular weight distribution was determined using a high-performance liquid chromatograph equipped with a diode array detector. The chromatographic column was a hydrophilic modified silica size exclusion column suitable for separating peptides. The mobile phase was a mixed solution of acetonitrile and water containing 0.1% trifluoroacetic acid by volume.

[0083] The flow rate of the liquid chromatography system was set to a constant 0.5 mL / min, the column temperature was maintained at 30℃, and the detection wavelength was set to 214 nm. Based on the retention time and logarithmic molecular weight calibration curve established in advance using standards such as cytochrome C, aprotinin, bacitracin, and glycylglycine, the peak area of ​​the sample was segmented and integrated using the chromatography workstation to calculate the relative mass percentage of short peptides with a molecular weight of less than 1000 Da.

[0084] The initial dry basis mass data of the substrate was retrieved from the production operation records of each batch. Combined with the weighing results of the final dry powder produced by spray drying, the total peptide solids yield of the entire preparation process of this batch was calculated using the basic mass balance formula.

[0085] The experimental results are shown in Table 3: Table 3: Test results of total peptide yield and short peptide mass percentage in each example and comparative example ; in conclusion: Figure 3 This is a comparison chart of the total yield and short peptide distribution ratio of various embodiments and comparative examples of the present invention. Figure 3 The horizontal axis corresponds to Examples 1 to 4 and Comparative Examples 1 to 4. The vertical axis on the left and the line connecting the solid dots in the figure correspond to the total polypeptide solids yield of each group. The vertical axis on the right and the line connecting the hollow rhombuses in the figure correspond to the relative mass percentage of short peptides less than 1000 Da in each group.

[0086] According to the data in Table 3, the total polypeptide solids yield of the example groups remained stable in the range of 72% to 80%, and the relative mass proportion of short peptides with a molecular weight of less than 1000 Da in the system reached more than 86%. In routine industrial polypeptide extraction, a technical bottleneck is often encountered in which it is difficult to simultaneously achieve the overall product yield and the enrichment degree of highly active microfragments. The underlying reason is that the target hydrophobic short peptides released by deep hydrolysis will expose more nonpolar residues due to thermal shock when undergoing unavoidable heat inactivation of enzyme units.

[0087] Comparative Example 1, which employed traditional autoclave enzyme inactivation methods, showed only a total yield of 42.3% and a short peptide distribution of 63.8%. This precipitous drop in data directly reflects the severe material loss caused by non-targeted heat-induced aggregation. During the high-temperature incubation period, the free, highly active small peptides rapidly underwent dense hydrophobic cross-linking and electrostatic flocculation with incompletely hydrolyzed macromolecular protein residues, forming macroscopically visible disordered co-precipitates that were subsequently discharged as waste from the liquid phase system during cleaning and centrifugation.

[0088] To prevent co-precipitation in harsh heating environments, we formulated a specific composite solubilizing buffer. Comparative Example 2, completely lacking this microscopic chemical protection, even with inline instantaneous heating designed to shorten heating time, still saw its overall product yield hover around 56%, failing to achieve a significant breakthrough. The dynamic hydration shell constructed by the guanidino group of L-arginine and citric acid provides a robust steric barrier for the short peptide during the heating phase, allowing this high-value fragment to escape the trappings of larger molecules.

[0089] Observing the test results of Comparative Example 3, it can be found that blindly mixing all chemical reagents into the reactor at room temperature did not achieve the expected macromolecular specificity removal effect. The premature introduction of sodium hexametaphosphate into the reaction system caused the macromolecular protein to begin local cross-linking before the extreme thermal denaturation and unfolding. This prematurely formed loose cross-linking network not only weakened the clarity of the subsequent precipitation separation boundary, but also non-selectively physically encapsulated some free short peptides in the early stage of formation.

[0090] In Comparative Example 4, because the negative pressure flash evaporation step was removed, the newly formed solid cross-linked material failed to undergo phase change-driven physical shrinkage during the slow cooling process in the tube at normal pressure. The loose, sponge-like precipitate grid still contained a large amount of target clear liquid rich in small peptides, resulting in a significant decline in the proportion of effective ingredients. By intervening in the molecular assembly sequence and physical quenching path, this process has demonstrated reliable engineering transformation value in avoiding the thermal loss of active ingredients.

[0091] Test Example 4: Test objective: To evaluate the effect of the preparation process on the targeted retention of the bioactivity of the characteristic antihypertensive peptides in the composition and its anti-enzymatic stability in a simulated in vitro digestion environment.

[0092] Experimental steps: Weigh 50 mg of each of the active peptide composition dry powders prepared in each example and comparative example, dissolve them in sodium borate buffer solution with a pH of 8.3 and dilute them sequentially to prepare peptide sample basic assay solutions with 6 different mass concentration gradients.

[0093] In addition, each group of dry powder samples was accurately weighed and placed in a pre-prepared simulated gastric juice model containing pepsin and a simulated intestinal juice model containing trypsin for sequential in vitro digestion. After digestion, the enzymatic reaction was terminated by boiling water bath and the supernatant was collected by centrifugation. The supernatant was also diluted with sodium borate buffer to prepare a series of digestion-grade assay solutions.

[0094] Add a specific volume of assay solution and commercial angiotensin-converting enzyme extract to a centrifuge tube, and pre-incubate in a 37°C water bath shaker for 10 minutes. Then add hippuryl-histyl-leucine substrate solution preheated to the same temperature to start the reaction, and continue the reaction for 30 minutes under light-protected and temperature-controlled conditions.

[0095] To terminate the enzymatic reaction, rapidly add 1.0 mol / L hydrochloric acid solution to the reaction system. Add an appropriate amount of ethyl acetate and shake vigorously to extract the hippuric acid product generated in the reaction. After standing and separating the layers, transfer the upper organic phase to a new glass test tube and place it in a vacuum centrifuge to evaporate the solvent.

[0096] The residue at the bottom of the test tube was completely reconstituted using deionized water. The chromatographic peak of hippuric acid was integrated and quantified using high-performance liquid chromatography at a wavelength of 228 nm. The inhibition percentage of angiotensin-converting enzyme by different concentrations of peptide solutions was calculated. The half-maximal inhibitory concentration (IC50) of each sample was obtained by nonlinear regression fitting.

[0097] The experimental results are shown in Table 4: Table 4: Results of half-maximal inhibitory concentration (IC50) determination of peptide products in each example and comparative example. ; in conclusion: Figure 4 This is a comparison chart of the blood pressure-lowering bioactivity indicators of various embodiments and comparative examples of the present invention. Figure 4 The horizontal axis corresponds to Examples 1 to 4 and Comparative Examples 1 to 4, and the vertical axis represents the half-maximal inhibitory concentration (IC50). Solid lines marked with solid circles represent the IC50 values ​​of baseline peptide samples without simulated digestion, while dashed lines marked with hollow triangles represent the IC50 values ​​of peptide samples after continuous simulated in vitro gastrointestinal digestion.

[0098] According to the data in Table 4, the IC50 values ​​of Examples 1 to 4 before digestion were all below 0.17 mg / mL. Although the values ​​increased slightly after in vitro simulated gastrointestinal digestion, they were generally controlled within the range of 0.22 mg / mL. Comparative Example 1, which used conventional autoclave heat inactivation, showed a sharp increase in its initial IC50 value to 0.745 mg / mL.

[0099] In in vitro activity evaluation systems, the antihypertensive effect is highly dependent on the specific spatial chelation between the C-terminal hydrophobic amino acid residues of the short peptide sequence and the zinc ions of the angiotensin-converting enzyme active center. In conventional heated environments lacking spatial conformational locking protection, peptides with highly active hydrophobic residues are easily entangled and folded together under thermodynamic drive, or are non-specifically adsorbed by the hydrophobic core of unhelicated macromolecular proteins. The key binding sites of short peptides that are physically embedded are severely masked, and their affinity for the target enzyme naturally declines after reconstitution.

[0100] Even with the introduction of a protective reagent in Comparative Example 3, the activity recovery of materials that did not follow the instantaneous cross-linking logic at a specific extreme point remained limited. The premature intervention of the polyphosphate cross-linking agent induced disordered electrostatic bridging of various molecular weight peptides within the system, resulting in a steric hindrance effect on the free-state characteristic active molecules by the formed loose network structure. The absence of the phase transition quenching step is directly reflected in the data of Comparative Example 4 as the irreversible loss of active components.

[0101] Instantaneous flash evaporation under negative pressure not only promotes the desorption and vaporization of volatile impurities in engineering, but also induces a violent liquid-phase cooling and rapid shrinkage effect of the precipitate structure. On a macroscopic level, this forcefully releases highly active short peptides from the swollen hydration layer of the cross-linked material into the supernatant. Combined with the data change trends before and after in vitro simulated digestion, the characteristic active composition obtained through multiple coupled interventions of process conditions maintains superior sequence resistance when faced with continuous cleavage and destruction by pepsin and trypsin.

[0102] The entire system avoids unnecessary side-chain oxidation damage and physical masking during the extraction process, fully maintaining the original compact structural characteristics of the natural hydrolysis release products. Relying on this process path of chemical steric hindrance locking combined with physical phase transition densification, the medicinal potential of high-value-added peptides is substantially and directionally preserved in the industrial extraction process.

[0103] Test Example 5: Test objective: To evaluate the engineering effects of phase change quenching and extreme point high shear crosslinking processes on eliminating mesoscopic macromolecular protein aggregates in the liquid phase, reducing concentration polarization, and delaying irreversible fouling of ultrafiltration membranes.

[0104] Experimental steps: A small-scale tangential cross-flow ultrafiltration membrane system with jacketed water bath temperature control was constructed. The membrane module was selected as a polyethersulfone spiral wound ultrafiltration membrane with an effective membrane area of ​​0.1 square meters and a molecular weight cutoff of 3000 Da. Before the test, the membrane was circulated and flushed with deionized water at a constant transmembrane pressure difference of 0.15 MPa for 30 minutes and the basic pure water flux was recorded.

[0105] The clear liquid collected after centrifugation in each embodiment and comparative example was transferred in equal volume to the feed tank of the ultrafiltration system. The feed pump was turned on and the system test operation temperature was maintained at 25°C. The concentrate reflux valve was adjusted to stabilize the transmembrane pressure difference at 0.15 MPa and the crossflow surface velocity was kept constant at 1.0 m / s.

[0106] After the system has been running for 10 minutes to drain the residual pure water in the pipeline and reach a hydrodynamically stable state, the initial feed liquid membrane flux is measured at the permeate outlet using a graduated cylinder and a stopwatch.

[0107] The above test parameters were maintained for continuous ultrafiltration operation for 120 minutes without any backwashing or mechanical cleaning of the membrane surface. The steady-state membrane flux of the permeate was measured again when the operation reached the end point.

[0108] The relative flux decay rate of the system under continuous operation was calculated based on the ratio of the steady-state membrane flux at the end of the operation to the initial feed liquid membrane flux. After the test, the feed liquid in the pipeline was drained and the system was cleaned with alkaline solution using standard chemical cleaning.

[0109] The experimental results are shown in Table 5: Table 5: Test results of separation efficiency and antifouling performance of ultrafiltration membranes in each example and comparative example ; in conclusion: Figure 5 This is a comparison chart of the separation efficiency of ultrafiltration membranes in various embodiments and comparative examples of the present invention. Figure 5 The horizontal axis corresponds to Examples 1 to 4 and Comparative Examples 1 to 4. The vertical axis on the left and the solid line marked with circles in the figure correspond to the steady-state membrane flux value after 120 minutes of continuous operation for each group. The vertical axis on the right and the dashed line marked with squares in the figure correspond to the relative flux decay rate value during the operation of each group.

[0110] According to the data in Table 5, the initial feed membrane flux of the example groups was at a high level, and after two hours of continuous cross-flow operation, the flux decline rate was controlled within 15%. Ultrafiltration separation at the end of peptide industrial extraction often encounters deterioration in filtration efficiency caused by membrane concentration polarization and pore blockage. Observing the data of Comparative Example 1 without pretreatment intervention, its final flux dropped to 16.4 L / m³. 2 ·h, the relative decay rate is close to 60%.

[0111] Large molecular weight proteins and various medium molecular weight aggregates that have not been fully denatured and precipitated inside the feed solution continue to migrate to the membrane interface under the driving force of operating pressure. These substances are very easy to undergo irreversible physical adsorption with polyethersulfone membrane material, forming a highly dense gel deposition layer on the cross-flow surface.

[0112] Comparative Example 3 attempted to mix the flocculant into the enzymatic hydrolysis system at room temperature. However, due to the lack of protein conformation unwinding and dissociation driven by thermal denaturation, the polyphosphate could only weakly bridge with a very small number of exposed charges on the substrate surface. The mechanical characteristics of the randomly generated flocs at low temperature are extremely loose. Under the high-speed vortex tearing and cross-flow channel shearing force of the ultrafiltration feed pump, they will dissociate again into fine fragments, which directly embed and block the micron-sized irregular membrane pores.

[0113] Even for the material in Comparative Example 4, which underwent high-shear crosslinking treatment at the extreme point of high-temperature denaturation, the attenuation rate still reached 33.9% after the vacuum flash quenching step was removed. Conventional heat exchange and slow cooling under normal pressure cannot induce macroscopic physical phase change. The solid crosslinked material still retains a large amount of water and exhibits a low-density, loose state. Such flocs are difficult to completely achieve solid-phase sedimentation in a horizontal screw centrifuge. The small suspended particles directly increase the feed load of the ultrafiltration system with the large amount of overflow of the clear liquid.

[0114] The process design of this embodiment accurately targets the brief time window during which proteins undergo thermal defolding, exposing their internal structure. A strong flow field forcibly intervenes in cross-linking and closely follows the phase transition process. The rapid latent heat absorption caused by flash degassing induces millisecond-level cold contraction in the liquid phase system, forcibly expelling free water from the loose polymer network and solidifying the structure into a shear-resistant, dense precipitate. This densification modification not only improves the thoroughness of slag removal during front-end centrifugation but also cuts off the source of particles that could potentially enter the ultrafiltration membrane pores and form deep-seated contamination, ensuring that the separation module maintains a high engineering permeation flux throughout its entire lifecycle.

Claims

1. A blood pressure-lowering protein active peptide composition, characterized in that, Made from raw materials comprising the following parts by weight: Protein substrate: 10-15 parts by weight; Deionized water: 100 parts by weight; Alkaline protease: 0.02-0.05 parts by weight; Compound solubilizing buffer: 5-10 parts by weight; Crosslinked aqueous solution: prepared from 10 parts by weight of deionized water and 0.15-0.25 parts by weight of sodium hexametaphosphate.

2. The antihypertensive protein active peptide composition according to claim 1, characterized in that, The solute in the composite solubilizing buffer is composed of L-arginine and anhydrous citric acid; The molar ratio of L-arginine to anhydrous citric acid in the composite solubilizing buffer is (2.2-2.8):1; The concentration of L-arginine in the composite solubilizing buffer is 1.0 mol / L.

3. The antihypertensive protein active peptide composition according to claim 1, characterized in that, The protein substrate is selected from casein or soy protein isolate; The initial addition temperature of the deionized water is 48-52℃; The cross-linked aqueous solution is prepared by mixing and dissolving the deionized water and the sodium hexametaphosphate.

4. The antihypertensive protein active peptide composition according to claim 1, characterized in that, The alkaline protease is an alkaline protease extracted by fermentation of Bacillus subtilis or an alkaline protease extracted by fermentation of Bacillus licheniformis. The alkaline protease has an enzyme activity of 200,000-400,000 U / g.

5. A method for preparing a blood pressure-lowering protein active peptide composition according to any one of claims 1-4, characterized in that, Includes the following steps: The protein substrate was added to preheated deionized water, and after adjusting the pH of the system, alkaline protease was added for constant temperature and dynamic pH adjustment of the stirring enzymatic hydrolysis. After the target degree of hydrolysis was reached, the enzymatic hydrolysate was obtained. The composite solubilizing buffer is added to the enzymatic hydrolysis solution, and after being stirred and mixed at a constant temperature, it is continuously fed into a heat exchanger for instantaneous heating to obtain a high-temperature solution. The crosslinking aqueous solution is injected into a high-shear mixing device located at the outlet of the heat exchanger. After instantaneous crosslinking with the high-temperature liquid, it is directly sprayed into a vacuum flash evaporation device to cause the mixed system to boil and vaporize and cool down instantly. After staying at the bottom of the tank, a modified crosslinking liquid is obtained. The denatured cross-linked liquid was subjected to mechanical solid-liquid separation, ultrafiltration cross-flow impurity removal, evaporation concentration and spray drying in sequence to obtain the final blood pressure lowering protein active peptide composition.

6. The method for preparing the antihypertensive protein active peptide composition according to claim 5, characterized in that, The composite solubilizing buffer solution is prepared in advance through the following steps: Anhydrous citric acid was added to deionized water and mechanically stirred at room temperature until completely dissolved to obtain an aqueous citric acid solution. Under continuous stirring, L-arginine was added to the citric acid aqueous solution, and stirring was continued until the solids were completely dissolved. Deionized water was added to bring the volume to a final level, yielding the composite solubilizing buffer solution.

7. The method for preparing the antihypertensive protein active peptide composition according to claim 5, characterized in that, The process of adding the protein substrate to preheated deionized water, adjusting the pH of the system, adding alkaline protease, and performing enzymatic hydrolysis with constant temperature and dynamic pH adjustment, until the target degree of hydrolysis is reached, specifically includes: The initial pH of the deionized water and protein substrate mixture was adjusted to 8.0 using an aqueous sodium hydroxide solution. The alkaline protease was added to the mixture, and enzymatic hydrolysis was carried out under constant temperature conditions with stirring. The sodium hydroxide aqueous solution was dynamically added during the enzymatic hydrolysis process to maintain the pH of the system at 8.0; When the degree of hydrolysis of the system reaches 15-18, stop adding the sodium hydroxide aqueous solution to obtain the enzymatic hydrolysate.

8. The method for preparing the antihypertensive protein active peptide composition according to claim 5, characterized in that, The step of adding the composite solubilizing buffer to the enzymatic hydrolysis solution, mixing it under constant temperature and stirring, and then continuously feeding it into a heat exchanger for instantaneous heating to obtain a high-temperature solution specifically includes: At a temperature of 48-52℃, stir the system containing the compound solubilizing buffer at a speed of 50-100 rpm for 15-20 minutes. The stirred material is continuously pumped into the shell and tube heat exchanger; The liquid is controlled to stay in the tube side of the shell-and-tube heat exchanger for 15-20 seconds, so that the temperature of the liquid at the outlet of the shell-and-tube heat exchanger reaches 93-97℃, thus obtaining the high-temperature liquid.

9. The method for preparing the antihypertensive protein active peptide composition according to claim 5, characterized in that, The process of injecting the crosslinking aqueous solution into a high-shear mixing device located at the outlet of the heat exchanger, instantaneously mixing and crosslinking it with the high-temperature liquid, and then directly spraying it into a vacuum flash evaporation device to cause the mixture to boil, vaporize, and cool down instantly, resulting in a denatured crosslinking liquid after it settles at the bottom of the tank, specifically includes: A pipeline-type static high-shear mixer is used as the high-shear mixing device, and the shear rate of mixing and crosslinking is controlled at 3000-5000 s⁻¹, and the mixing and crosslinking time is 1.0-2.0 seconds. The absolute pressure of the vacuum flash evaporation equipment is controlled to be constant at 0.02-0.03 MPa; The mixture is cooled to 58-62°C within 1-3 seconds and remains at the bottom of the vacuum flash evaporator for 3-5 minutes.

10. The method for preparing the antihypertensive protein active peptide composition according to claim 5, characterized in that, The denatured cross-linked feed solution is subjected to mechanical solid-liquid separation, ultrafiltration cross-flow impurity removal, evaporation concentration, and spray drying in sequence to obtain the final antihypertensive protein active peptide composition, which specifically includes: The modified crosslinked liquid is continuously fed into a horizontal screw discharge centrifuge, where mechanical solid-liquid separation is performed at a separation factor of 3000-5000g, and the supernatant is collected. The supernatant is pumped into the ultrafiltration membrane module to perform ultrafiltration cross-flow impurity removal, and the permeate is collected; The permeate is fed into a mechanical vapor recompression evaporator for evaporation and concentration, and the concentration is controlled to contain 20-30 parts by weight of solids per 100 parts by weight of concentrate. The concentrated material is spray-dried to obtain the final blood pressure-lowering protein active peptide composition dry powder.

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