Black fungus antihypertensive peptide culture medium composition based on selenium-enriched yeast protein, liquid fermentation method and antihypertensive application of black fungus antihypertensive peptide culture medium composition

Abstract

The invention relates to the technical field of biological medicine and food fermentation, in particular to a black fungus antihypertensive peptide culture medium composition based on selenium-enriched yeast protein, a liquid fermentation method and antihypertensive application of the black fungus antihypertensive peptide culture medium composition. The culture medium comprises selenium-enriched yeast protein, glucose, fermented soybean water extract dregs, inorganic salt and trace elements, the high-activity antihypertensive peptide is obtained by optimizing a liquid fermentation and separation purification process, the ACE inhibition IC50 value of the high-activity antihypertensive peptide is smaller than 50 micrograms per milliliter, and the high-activity antihypertensive peptide can be compounded with a hawthorn extract and the like to prepare an oral preparation and has a remarkable antihypertensive function.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedicine and food fermentation, specifically a black fungus antihypertensive peptide culture medium composition based on selenium-enriched yeast protein, a liquid fermentation method, and its application in the field of blood pressure reduction. Background Technology

[0002] As a precious product of the edible fungus kingdom, black fungus is a fungus that can be used for both food and medicine. Its medicinal value has attracted widespread attention from consumers. It has the effects of helping to lower blood pressure, regulate blood lipids, and prevent coagulation, and has potential benefits for hypertension, arteriosclerosis, hyperlipidemia, and cardiovascular and cerebrovascular diseases.

[0003] Black fungus mycelium has advantages such as controllable growth cycle, strong adaptability to liquid fermentation, and rich metabolites. By optimizing the culture medium and fermentation process, it can be used to synthesize bioactive peptides, which can help increase the content of target components, enhance product stability, and promote industrialization. Summary of the Invention

[0004] The purpose of this invention is to provide a black fungus antihypertensive peptide culture medium composition based on selenium-enriched yeast protein, a liquid fermentation method, and its application in lowering blood pressure.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a culture medium composition for black fungus antihypertensive peptides based on selenium-enriched yeast protein, the culture medium composition comprising selenium-enriched yeast protein, glucose, light soybean water extract residue, potassium dihydrogen phosphate, magnesium sulfate, calcium carbonate and trace element solution.

[0006] Furthermore, in a preferred embodiment of the present invention, by mass percentage, the content of selenium-enriched yeast protein is 1% to 5%, glucose is 2% to 6%, fermented soybean water extract residue is 0.5% to 2%, potassium dihydrogen phosphate is 0.1% to 0.5%, magnesium sulfate is 0.05% to 0.2%, calcium carbonate is 0.1% to 0.5%, trace element solution is 0.1% to 0.5%, and the balance is water.

[0007] Furthermore, in a preferred embodiment of the present invention, the selenium-enriched yeast protein is a brewer's yeast protein obtained through biotransformation, with an organic selenium content of 1000 mg to 2000 mg per kilogram and a protein content of not less than 70%, wherein the selenium-containing amino acids exist in the form of selenocysteine ​​and selenomethionine.

[0008] Furthermore, in a preferred embodiment of the present invention, the residue of the light fermented soybean water extract is the solid residue after the extraction of soluble components by the water extraction process. After being treated by air jet milling, the particle size is 40 to 60 mesh, and its components include β-glucan, isoflavones and free amino acids.

[0009] Furthermore, in a preferred embodiment of the present invention, the trace element solution is prepared from ferrous citrate, manganese sulfate, zinc sulfate and copper sulfate, wherein the iron concentration is 50 mg to 80 mg per liter, the manganese concentration is 10 mg to 20 mg per liter, the zinc concentration is 5 mg to 15 mg per liter, and the copper concentration is 0.5 mg to 2 mg per liter.

[0010] Furthermore, in a preferred embodiment of the present invention, by mass percentage, the content of selenium-enriched yeast protein is 3%, glucose is 4%, light soybean water extract residue is 1%, potassium dihydrogen phosphate is 0.3%, magnesium sulfate is 0.1%, calcium carbonate is 0.3%, trace element solution is 0.3%, and the remainder is water.

[0011] Secondly, the present invention provides a liquid fermentation method for black fungus antihypertensive peptides, comprising: Add 1% to 5% selenium-enriched yeast protein, 2% to 6% glucose, 0.5% to 2% fermented soybean water extract residue, 0.1% to 0.5% potassium dihydrogen phosphate, 0.05% to 0.2% magnesium sulfate, 0.1% to 0.5% calcium carbonate, and 0.1% to 0.5% trace element solution to water and stir well. Adjust the pH to 6.0 to 6.5, sterilize at 121℃ for 20 to 30 minutes, and obtain a liquid fermentation medium after cooling. Incubate black fungus on PDA slant medium at 25℃ to 28℃ for 7 to 10 days to obtain activated strains. Inoculate the activated strains into a seed culture medium containing 3% glucose, 0.5% fermented soybean water extract residue, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, and the remainder being water. Seed culture was obtained by culturing at 25℃ to 28℃ and shaking at 150r / min to 200r / min for 3 to 5 days. The seed culture was then inoculated into the liquid fermentation medium at an inoculum rate of 5% to 10%, and fermented at 25℃ to 28℃ and shaking at 150r / min to 200r / min for 7 to 10 days, maintaining a dissolved oxygen concentration of not less than 40% and a pH of 6.0 to 6.5. After fermentation, the fermentation broth was centrifuged at 4000r / min for 20 minutes, and the supernatant was collected. The supernatant was then ultrafiltered to retain the fraction with a molecular weight cutoff of 1000Da to 3000Da. The fraction was then separated by chromatography through a strong cation exchange resin column and a cross-linked dextran gel column, and the target elution peak fraction was collected. After freeze-drying, the black fungus antihypertensive peptide was obtained.

[0012] Further, in a preferred embodiment of the present invention, the liquid fermentation medium contains 3% selenium-enriched yeast protein, 4% glucose, 1% fermented soybean water extract residue, 0.3% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.3% calcium carbonate, 0.3% trace element solution, and the remainder is water; the seed culture inoculum is 8%; the fermentation temperature is 26°C, and the shaking speed is 180 r / min; dissolved oxygen is controlled by adjusting the aeration rate and stirring speed from 150 r / min to 200 r / min via an online dissolved oxygen electrode; pH is controlled by adding 0.5 mol / L ammonia or 0.1 mol / L lactic acid solution via a peristaltic pump to maintain it at 6.0 to 6.5; ultrafiltration uses a polyethersulfone flat sheet membrane module with an operating pressure of 0.2 MPa to 0.3 MPa; ion exchange chromatography uses SP Sepharose Fast Flow resin, with the sample pH adjusted to 4.0 before loading, and the elution gradient being a linear gradient of 0 mol / L to 1 mol / L NaCl; gel filtration chromatography uses Sephadex. G-25 medium, mobile phase is 0.1 mol / L phosphate buffer, flow rate is 1 mL / min to 2 mL / min.

[0013] Further, in a preferred embodiment of the present invention, the liquid fermentation culture medium contains 3% selenium-enriched yeast protein, 4% glucose, 1% fermented soybean water extract residue, 0.3% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.3% calcium carbonate, 0.3% trace element solution, and the remainder is water; the seed culture inoculum is 8%; the fermentation temperature is 26℃, the shaking speed is 180 r / min; dissolved oxygen is maintained above 40%; pH is maintained between 6.0 and 6.5; the fermentation cycle is 8 days; the centrifugation parameters are 4000 r / min for 20 minutes; the ultrafiltration molecular weight cutoff is 2000 Da; the ion exchange chromatography elution retention time is 35 to 40 minutes; the molecular weight corresponding to the gel filtration chromatography elution peak is approximately 2000 Da; the obtained antihypertensive peptide, after freeze-drying, has a water content of less than 5%, is sealed and nitrogen-filled, and stored at 4℃ in the dark.

[0014] Thirdly, the present invention also provides a method for applying the black fungus antihypertensive peptide, which includes: mixing the black fungus antihypertensive peptide with hawthorn extract, cassia seed polysaccharide, and buckwheat flavonoids in a mass ratio of 1:0.5:0.3:0.2 to prepare an oral preparation; the total flavonoid content of the hawthorn extract is not less than 20%, the molecular weight of the cassia seed polysaccharide is 50kDa to 100kDa, and the rutin content in the buckwheat flavonoids is not less than 80%; the mass fraction of the black fungus antihypertensive peptide in the oral preparation is 0.1% to 1%; the purity of the black fungus antihypertensive peptide is not less than 98%, and the ACE inhibition IC50 value is less than 50μg / mL.

[0015] Furthermore, in a preferred embodiment of the present invention, the black fungus antihypertensive peptide is mixed with hawthorn extract, cassia seed polysaccharide, and buckwheat flavonoids in a mass ratio of 1:0.5:0.3:0.2 and then filled into hard capsule shells, each capsule containing 5mg of black fungus antihypertensive peptide; or it is dissolved in sterile physiological saline, filtered through a 0.22μm microporous membrane for sterilization, and then filled into ampoules, each 1mL. Detailed Implementation

[0016] The specific embodiments of the present invention are as follows: The liquid fermentation process for the black fungus antihypertensive peptide of this invention includes the following steps: first, seed culture is carried out; then, a liquid fermentation medium is prepared; next, the activated strain is inoculated into the medium for fermentation; after fermentation, the fermentation broth is centrifuged; the resulting supernatant is subjected to ultrafiltration and then sequentially passed through ion exchange chromatography and gel filtration chromatography to finally obtain the target product, which is then freeze-dried. The above steps are sequentially connected, with the output of the previous step serving as the input of the next, forming a complete continuous preparation path.

[0017] In the seed culture stage, black fungus strains preserved on PDA slant medium were selected and statically cultured at a constant temperature of 25℃ to 28℃ for 7 to 10 days to allow the mycelium to grow fully and complete activation. Subsequently, the activated mycelial blocks were transferred to Erlenmeyer flasks containing seed culture medium, which consisted of 3% glucose, 0.5% light soybean water extract residue, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, and the remainder water. The flasks were placed in a shaker and cultured at a shaking speed of 150 r / min to 200 r / min and a temperature of 25℃ to 28℃ for 3 to 5 days to obtain a uniformly turbid seed liquid with obvious mycelial ball structure. This seed liquid was used as the inoculum source for subsequent liquid fermentation.

[0018] The preparation process of the liquid fermentation medium is as follows: Weigh out 3% selenium-enriched yeast protein, 4% glucose, 1% fermented soybean water extract residue, 0.3% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.3% calcium carbonate, 0.3% trace element solution, and the remainder is water; among them, the selenium-enriched yeast protein is brewer's yeast protein obtained through biotransformation, with an organic selenium content of 1000 mg to 2000 mg per kilogram, a protein content of not less than 70%, and selenium-containing amino acids existing in the form of selenocysteine ​​and selenomethionine; the fermented soybean water extract residue is the solid residue after the water extraction process for soluble components, which is treated by air jet milling and the particle size is controlled within the range of 40 to 60 mesh, and its components include β-glucan, isoflavones and free amino acids; the trace element solution consists of ferrous citrate, manganese sulfate, ... Zinc sulfate and copper sulfate were dissolved in deionized water in a specific ratio to prepare a solution with iron concentrations of 50-80 mg / L, manganese concentrations of 10-20 mg / L, zinc concentrations of 5-15 mg / L, and copper concentrations of 0.5-2 mg / L. These components were then added sequentially to a stainless steel fermenter, and the agitator was turned on and stirred at 300 rpm for 30 minutes to ensure complete dissolution and homogeneity. The pH of the system was then monitored using a pH meter and adjusted to 6.0-6.5 by adding 1 mol / L sodium hydroxide or hydrochloric acid solution. After adjustment, all vents in the fermenter were closed, and a steam generator was connected. The system was sterilized at 121°C for 20-30 minutes. After sterilization, the system was allowed to cool naturally to room temperature for later use.

[0019] During inoculation and fermentation, the aforementioned seed culture was pumped into the sterilized and cooled liquid fermentation medium at an inoculation rate of 8% by volume. After inoculation, the fermenter control system was started, the fermentation temperature was set to 26°C, the oscillation speed was set to 180 r / min, and the dissolved oxygen concentration was monitored in real time by an online dissolved oxygen electrode. When the dissolved oxygen was lower than 40%, the aeration rate and stirring speed were automatically adjusted from 150 r / min to 200 r / min to maintain the dissolved oxygen at no less than 40%. At the same time, a peristaltic pump was connected to an ammonia storage tank of 0.5 mol / L and a lactic acid storage tank of 0.1 mol / L, and the corresponding solutions were automatically added according to the pH online detection signal to keep the pH of the system stable in the range of 6.0 to 6.5. The entire fermentation cycle lasted for 8 days. During this period, samples were taken daily to observe the mycelial morphology and metabolic state to ensure that there was no contamination by other microorganisms and that the fermentation process was normal.

[0020] After fermentation, all fermentation broth was transferred to a centrifuge and centrifuged at 4000 r / min for 20 minutes. The supernatant was collected and the precipitate was discarded. The supernatant was then introduced into an ultrafiltration system, using a polyethersulfone flat sheet membrane module with a molecular weight cutoff of 2000 Da. Cross-flow filtration was performed at an operating pressure of 0.2 MPa to 0.3 MPa to retain components with a molecular weight less than 2000 Da in the permeate and remove macromolecular impurities and undegraded proteins.

[0021] The ultrafiltration fraction was fed into an ion exchange chromatography unit using SP Sepharose Fast Flow strong cation exchange resin with a column volume of 20 mL. Before loading, the sample pH was adjusted to 4.0 to match the optimal binding conditions of the resin. Elution was performed using a linear gradient of 0 mol / L to 1 mol / L NaCl at a flow rate of 1.5 mL / min. During elution, the absorbance at 280 nm was monitored in real time using a UV detector, and the elution curve was recorded. The target elution peak appeared in the retention time range of 35 to 40 minutes, corresponding to an active peptide rich in positively charged amino acid residues.

[0022] Subsequently, the target eluent collected by ion exchange chromatography was introduced into a gel filtration chromatography column. The packing material was Sephadex G-25 cross-linked dextran gel, and the column volume was 30 mL. The mobile phase was 0.1 mol / L phosphate buffer at pH 6.5, and the flow rate was set to 1.5 mL / min. The elution process was monitored by a UV detector. The molecular weight corresponding to the target elution peak was approximately 2000 Da, indicating that the component was mainly composed of short-chain peptides, which is consistent with the expected characteristics of antihypertensive peptide molecules.

[0023] After two-step chromatographic purification, the target component was transferred to a freeze dryer and pre-frozen at -50°C for 4 hours. Then, it was sublimated and dried under vacuum of ≤10Pa and cold trap temperature of -60°C for 24 hours. After drying, a white, loose powdery product was obtained, which is the black fungus antihypertensive peptide. Its water content was less than 5% as determined by Karl Fischer method. After sealing and filling with nitrogen, it was stored at 4°C in the dark.

[0024] To further verify the application performance of the obtained black fungus antihypertensive peptide, it was formulated into different dosage forms: In the preparation of oral formulations, the black fungus antihypertensive peptide was mixed with hawthorn extract, cassia seed polysaccharide, and buckwheat flavonoids at a mass ratio of 1:0.5:0.3:0.2; wherein the total flavonoid content of hawthorn extract was not less than 20%, the molecular weight of cassia seed polysaccharide was 50kDa to 100kDa, and the rutin content in buckwheat flavonoids was not less than 80%; the mass fraction of black fungus antihypertensive peptide in the mixture was controlled at 0.1% to 1%, and the remainder was supplemented by excipients; the mixed powder was filled into No. 0 hard capsule shells, and each capsule contained 5mg of black fungus antihypertensive peptide.

[0025] Throughout the entire process, the various equipment units are physically connected and logically linked through pipelines, pumps, valves, and control systems: the shaker used for seed culture is connected to the liquid fermentation tank via a sterile transfer pipeline to ensure a closed and contamination-free inoculation process; the centrifuge outlet is connected to the inlet of the ultrafiltration system via a silicone hose; the permeate outlet of the ultrafiltration system is transported to the inlet of the ion exchange chromatography column via a peristaltic pump; the outlet of the ion exchange chromatography column is then switched to the inlet of the gel filtration chromatography column via a three-way valve; finally, the collection tube of the gel filtration chromatography is directly connected to the sample tray of the freeze dryer; all key parameters such as temperature, pH value, dissolved oxygen concentration, oscillation speed, and fermentation time are centrally collected and controlled by the PLC control system to ensure process stability and reproducibility.

[0026] To enable those skilled in the art to fully understand and implement this invention, the specific implementation principles of this invention are further supplemented below with a specific application scenario.

[0027] In the industrial preparation of black fungus antihypertensive peptides, the entire process is based on continuous and closed-loop operation, with each unit achieving efficient synergy through physical connection and logical control. First, in the seed culture stage, black fungus strains preserved on PDA slant are placed in a constant temperature environment of 25℃ to 28℃ for 7 to 10 days. This process activates mycelial metabolic activity and restores its growth capacity. Subsequently, the strains are transferred to a seed culture medium containing 3% glucose and 0.5% fermented soybean water extract residue, and cultured at a shaking rate of 150 rpm to 200 rpm for 3 to 5 days, forming dense and uniformly dispersed mycelial balls. The principle behind this operation is that moderate shear force promotes mycelial fragmentation and proliferation, while the free amino acids and β-glucan in the fermented soybean water extract residue act as nitrogen sources and inducing factors, synergistically enhancing cell vitality and laying the foundation for subsequent high-density fermentation.

[0028] In the liquid fermentation medium preparation stage, components such as 3% selenium-enriched yeast protein, 4% glucose, and 1% fermented soybean water extract residue were precisely weighed according to their mass percentages. The selenium-enriched yeast protein contains selenocysteine ​​and selenomethionine, which not only provide a high-quality nitrogen source but also enhance the antioxidant capacity of the cells by participating in glutathione peroxidase synthesis through selenium, thereby prolonging the peak metabolic period. The fermented soybean water extract residue, after being pulverized to 40–60 mesh by air jet milling, retains isoflavones and β-glucan, which can act as secondary metabolic inducers, directionally promoting the synthesis of ACE inhibitory peptides. Calcium carbonate buffers pH fluctuations, preventing the accumulation of organic acids that could lead to premature fermentation termination. All components were homogenized and dissolved in a stainless steel tank by stirring at 300 rpm for 30 minutes, and then sterilized by steam to thoroughly eliminate interference from other microorganisms, ensuring a pure culture fermentation environment.

[0029] During the inoculation and fermentation stages, the seed culture was pumped into a sterilized fermenter at an 8% volume ratio. The PLC control system was activated, and the temperature was set to 26℃ and the oscillation speed to 180 r / min. Dissolved oxygen concentration was monitored in real-time by online electrodes. When it fell below 40%, the system automatically increased the aeration rate and adjusted the stirring speed to 150–200 r / min. The principle behind this is that *Auricularia auricula-judae* mycelium is an aerobic fungus; maintaining dissolved oxygen ≥40% ensures efficient electron transport in the respiratory chain, preventing the metabolism from shifting to ethanol fermentation due to hypoxia and inhibiting peptide synthesis. Simultaneously, the pH was dynamically maintained at 6.0–6.5 by adding 0.5 mol / L ammonia or 0.1 mol / L lactic acid via a peristaltic pump. This range not only matches the optimal pH for *Auricularia auricula-judae* growth but also promotes protease activity expression, thereby facilitating the hydrolysis of endogenous proteins into small peptides. The entire fermentation cycle lasted 8 days. During this period, the mycelium continuously secreted extracellular proteases, directionally degrading large proteins in selenium-enriched yeast proteins into short peptides with ACE inhibitory activity.

[0030] After fermentation, the fermentation broth was centrifuged to remove mycelia and insoluble residues, and the supernatant was introduced into an ultrafiltration system. A polyethersulfone membrane with a molecular weight cutoff of 2000 Da was used for cross-flow filtration at a pressure of 0.2–0.3 MPa. The separation principle is based on the molecular sieve effect: the target pressure-lowering peptides have a molecular weight concentration of 1000–2000 Da, which can pass through the membrane pores smoothly, while undegraded large molecular proteins and polysaccharide complexes are retained, thereby initially enriching the active components.

[0031] Subsequently, the ultrafiltration permeate entered the ion exchange chromatography unit using SP Sepharose Fast Flow strong cation exchange resin. Because the antihypertensive peptides from black fungus are rich in basic amino acid residues such as lysine and arginine, they carry a positive charge at pH 4.0 and can specifically bind to the sulfonic acid groups of the resin. During elution, a linear gradient of 0–1 mol / L NaCl was used to gradually shield against electrostatic interactions, allowing peptides with different charge densities to desorb sequentially. The target peak appeared at 35–40 min, corresponding to the component with high ACE inhibitory activity. This step achieved precise separation based on charge differences.

[0032] The collected eluent was then subjected to gel filtration chromatography using Sephadex G-25 media, and secondary purification was performed based on the principle of molecular size exclusion. The mobile phase was 0.1 mol / L phosphate buffer at pH 6.5. The target elution peak corresponded to a molecular weight of approximately 2000 Da, consistent with the typical molecular characteristics of angiotensin-converting enzyme (ACE) inhibitory peptides.

[0033] Finally, the purified peptide solution was freeze-dried to obtain a stable powder. Pre-freezing to -50°C allowed for complete water crystallization, followed by sublimation under ≤10Pa vacuum and a -60°C cold trap, effectively preserving the peptide's spatial conformation and biological activity. The resulting product had a water content of <5%, facilitating long-term storage.

[0034] At the application level, the antihypertensive peptide from black fungus is compounded with hawthorn extract, cassia seed polysaccharide, and buckwheat flavonoids in a ratio of 1:0.5:0.3:0.2 to form an oral preparation. Its synergistic mechanism lies in the following: hawthorn flavonoids can dilate coronary arteries, cassia seed polysaccharide regulates lipid metabolism, buckwheat flavonoids improve microcirculation, and the antihypertensive peptide from black fungus directly inhibits ACE activity, blocking the conversion of angiotensin I to II, thereby regulating blood pressure at multiple targets.

[0035] From seed culture to freeze drying, each piece of equipment is integrated with a PLC central controller through sterile pipelines. Key parameters such as temperature, pH, dissolved oxygen, rotation speed, and time are all subject to closed-loop feedback control to ensure batch-to-batch consistency and meet GMP production requirements.

[0036] All content not described in detail in the specification is prior art known to those skilled in the art and will not be described further.

[0037] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium composition for black fungus antihypertensive peptides based on selenium-enriched yeast protein, characterized in that, The culture medium composition comprises selenium-enriched yeast protein, glucose, fermented soybean water extract residue, potassium dihydrogen phosphate, magnesium sulfate, calcium carbonate, and trace element solution. By mass percentage, the selenium-enriched yeast protein is 1% to 5%, glucose is 2% to 6%, fermented soybean water extract residue is 0.5% to 2%, potassium dihydrogen phosphate is 0.1% to 0.5%, magnesium sulfate is 0.05% to 0.2%, calcium carbonate is 0.1% to 0.5%, trace element solution is 0.1% to 0.5%, and the balance is water.

2. The culture medium composition for black fungus antihypertensive peptides according to claim 1, characterized in that, The selenium-enriched yeast protein is a brewer's yeast protein obtained through biotransformation, with an organic selenium content of 1000 mg to 2000 mg per kilogram and a protein content of not less than 70%, wherein the selenium-containing amino acids exist in the form of selenocysteine ​​and selenomethionine.

3. The black fungus antihypertensive peptide culture medium composition according to claim 1, characterized in that, The residue of the light fermented soybean water extract is the solid residue after the water extraction process extracts soluble components. After being treated by air jet milling, the particle size is 40 to 60 mesh. Its components include β-glucan, isoflavones and free amino acids.

4. The black fungus antihypertensive peptide culture medium composition according to claim 1, characterized in that, The trace element solution is prepared from ferrous citrate, manganese sulfate, zinc sulfate and copper sulfate, wherein the iron concentration is 50 mg to 80 mg per liter, the manganese concentration is 10 mg to 20 mg per liter, the zinc concentration is 5 mg to 15 mg per liter, and the copper concentration is 0.5 mg to 2 mg per liter.

5. The black fungus antihypertensive peptide culture medium composition according to claim 1, characterized in that, By mass percentage, the composition is 3% selenium-enriched yeast protein, 4% glucose, 1% fermented soybean water extract residue, 0.3% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.3% calcium carbonate, 0.3% trace element solution, and the remainder is water.

6. A method for liquid fermentation of black fungus antihypertensive peptides, characterized in that, Includes the following steps: Add 1% to 5% selenium-enriched yeast protein, 2% to 6% glucose, 0.5% to 2% fermented soybean water extract residue, 0.1% to 0.5% potassium dihydrogen phosphate, 0.05% to 0.2% magnesium sulfate, 0.1% to 0.5% calcium carbonate, and 0.1% to 0.5% trace element solution to water and stir evenly. Adjust the pH value to 6.0 to 6.5, sterilize at 121℃ for 20 to 30 minutes, and obtain liquid fermentation culture medium after cooling. Activated strains of black fungus were obtained by culturing black fungus on PDA slant medium at 25°C to 28°C for 7 to 10 days. The activated bacterial strain was inoculated into a seed culture medium containing 3% glucose, 0.5% of the residue from the water extract of fermented soybean, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, and the remainder being water. The seed culture was obtained by incubating the culture at 25°C to 28°C and shaking at 150 r / min to 200 r / min for 3 to 5 days. The seed culture was inoculated into the liquid fermentation medium at an inoculation rate of 5% to 10%, and fermented for 7 to 10 days under the conditions of 25°C to 28°C and shaking at 150 r / min to 200 r / min, while maintaining a dissolved oxygen concentration of not less than 40% and a pH value of 6.0 to 6.

5. After fermentation, centrifuge the fermentation broth at 4000 r / min for 20 minutes and collect the supernatant. The supernatant was subjected to ultrafiltration to retain components with molecular weights from 1000 Da to 3000 Da; The target elution peak components were then separated by chromatography using a strong cation exchange resin column and a cross-linked dextran gel column, and the elution peak components were collected and freeze-dried to obtain the black fungus antihypertensive peptide.

7. The method for liquid fermentation of black fungus antihypertensive peptides according to claim 6, characterized in that, The liquid fermentation medium contained 3% selenium-enriched yeast protein, 4% glucose, 1% fermented soybean water extract residue, 0.3% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.3% calcium carbonate, 0.3% trace element solution, and the remainder was water; the seed culture inoculum was 8%; the fermentation temperature was 26℃, and the shaking speed was 180 r / min; dissolved oxygen was controlled by adjusting the aeration rate and stirring speed from 150 r / min to 200 r / min via an online dissolved oxygen electrode; pH was controlled by adding 0.5 mol / L ammonia or 0.1 mol / L lactic acid solution via a peristaltic pump to maintain it at 6.0 to 6.5; ultrafiltration used a polyethersulfone flat sheet membrane module with an operating pressure of 0.2 MPa to 0.3 MPa; ion exchange chromatography used SP Sepharose Fast Flow resin, with the sample pH adjusted to 4.0 before loading, and an elution gradient of 0 mol / L to 1 mol / L NaCl linear gradient; gel filtration chromatography used Sephadex. G-25 medium, mobile phase is 0.1 mol / L phosphate buffer, flow rate is 1 mL / min to 2 mL / min.

8. The method for liquid fermentation of black fungus antihypertensive peptides according to claim 6, characterized in that, The liquid fermentation medium contains 3% selenium-enriched yeast protein, 4% glucose, 1% fermented soybean water extract residue, 0.3% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.3% calcium carbonate, 0.3% trace element solution, and the remainder is water; the seed liquid inoculation amount is 8%; the fermentation temperature is 26℃, the shaking speed is 180 r / min; and the dissolved oxygen is maintained above 40%. The pH was maintained between 6.0 and 6.5; the fermentation period was 8 days; the centrifugation parameters were 4000 r / min for 20 minutes; the molecular weight cutoff for ultrafiltration was 2000 Da; the elution retention time for ion exchange chromatography was 35 to 40 minutes; the molecular weight corresponding to the elution peak in gel filtration chromatography was approximately 2000 Da; the obtained antihypertensive peptides were freeze-dried to a water content of less than 5%, sealed and nitrogen-filled, and stored at 4°C in the dark.

9. A method for applying a black fungus antihypertensive peptide, characterized in that, The black fungus antihypertensive peptide is mixed with hawthorn extract, cassia seed polysaccharide, and buckwheat flavonoids in a mass ratio of 1:0.5:0.3:0.2 to prepare an oral preparation; the total flavonoid content of the hawthorn extract is not less than 20%, and the molecular weight of the cassia seed polysaccharide is 50kDa to 100kDa; the mass fraction of the black fungus antihypertensive peptide in the oral preparation is 0.1% to 1%; the purity of the black fungus antihypertensive peptide is not less than 98%, and the ACE inhibition IC50 value is less than 50μg / mL.

10. The method of applying the black fungus antihypertensive peptide according to claim 9, characterized in that, The black fungus antihypertensive peptide is mixed with hawthorn extract, cassia seed polysaccharide and buckwheat flavonoids in a mass ratio of 1:0.5:0.3:0.2 and then filled into hard capsules. Each capsule contains 5mg of black fungus antihypertensive peptide.