Preparation method of micrococcus parasuis protein with high ACE (angiotensin converting enzyme) inhibitory activity and micrococcus parasuis protein

Abstract

The invention provides a preparation method of a pseudococcus protein with high ACE (angiotensin converting enzyme) inhibitory activity and the pseudococcus protein, and relates to the field of food protein processing. Comprising the following steps: providing a micrococcus sample, mixing the micrococcus sample with a buffer solution, adjusting the pH value to 8-10, and carrying out wall breaking treatment to obtain a mixed solution; mixing the mixed solution with a compound enzyme, performing enzymolysis, performing enzyme deactivation, and performing centrifugation so as to obtain a supernatant, the compound enzyme comprising cellulase and alkaline protease; the supernatant is subjected to a salting-out reaction to separate out micronannochloropsis protein, the separated-out micronannochloropsis protein is subjected to aftertreatment, and the micronannochloropsis protein with the high ACE inhibitory activity is obtained.According to the method, a micronannochloropsis sample serves as a raw material, pollution is avoided, and the production cost of the micronannochloropsis protein can be remarkably reduced; no toxic substance is used in the whole process, and the method is safer when applied to the field of food processing.

Description

Technical Field

[0001] This invention relates to the field of food protein processing technology, and in particular to a method for preparing a micrococcus protein with high ACE inhibitory activity and the micrococcus protein itself. Background Technology

[0002] With increasing global emphasis on sustainable development and environmental protection, the extraction of high-value-added products from microalgal resources has become a research hotspot. *Nannochloropsis gaditana*, an important microalgal resource, has significant applications in biofuels and ω-3 polyunsaturated fatty acids due to its efficient oil production characteristics. However, the proteins in *Nannochloropsis gaditana* also have high value and can be used in the food, feed, and health product industries. Therefore, research on its protein extraction technology is of great significance. Optimizing and improving protein extraction methods not only helps to increase protein extraction efficiency but also provides high-quality raw materials for related industries.

[0003] Protein constitutes a significant portion of the dry matter of *Chlorella pseudocarpa*, with a protein content of 35%-40% compared to other protein sources. Furthermore, most plant proteins lack sufficient essential amino acids; for example, corn lacks tryptophan and lysine, cereals lack lysine, and legumes lack methionine. Therefore, *Chlorella pseudocarpa* can be considered an ideal protein source. However, currently, *Chlorella pseudocarpa* is primarily used for EPA lipid extraction, resulting in a substantial waste of protein resources. To address these technical challenges, scholars both domestically and internationally have conducted extensive research.

[0004] Currently, proteins are prepared from *Micrococcus pseudochlorella* using freeze-thaw salt precipitation, enzymatic extraction-assisted methods, and alkali-soluble acid precipitation methods. However, the prepared proteins have the following shortcomings: First, the defatted *Micrococcus pseudochlorella* proteins have poor water solubility and lose functional properties such as emulsifying and foaming properties. Second, the dissolved proteins are not effectively extracted. Third, the preparation methods are relatively complex and the production cost is high. Summary of the Invention

[0005] The main objective of this invention is to propose a method for preparing a microchlorella protein with high ACE inhibitory activity and the microchlorella protein itself, aiming to improve the extraction efficiency of the microchlorella protein while simplifying the operation.

[0006] To achieve the above objectives, this invention proposes a method for preparing a microphyte-like protein with high ACE inhibitory activity, comprising the following steps:

[0007] S10. Provide a sample of *Micrococcus pseudocarpa*, mix the sample with a buffer solution, adjust the pH value to 8-10, and then perform cell wall disruption treatment to obtain a mixed solution.

[0008] S20. The mixture is mixed with the complex enzyme, enzymatically hydrolyzed, enzyme inactivated, and centrifuged to obtain a supernatant, wherein the complex enzyme includes cellulase and alkaline protease.

[0009] S30. The supernatant is subjected to a salting-out reaction to precipitate Micrococcus pseudocarpa protein. The precipitated Micrococcus pseudocarpa protein is then post-processed to obtain Micrococcus pseudocarpa protein with high ACE inhibitory activity.

[0010] In one embodiment, in step S10, the *Micrococcus pseudocarpa* sample includes *Micrococcus pseudocarpa* powder or wet algae.

[0011] In one embodiment, in step S10:

[0012] The buffer solution includes phosphate buffer, and the ratio of the *Micrococcus pseudocarpa* sample to the buffer solution is: 15-20 mL of phosphate buffer solution is added for every 1 g of *Micrococcus pseudocarpa* sample.

[0013] In one embodiment, in step S10, the conditions for the cell wall disruption treatment include:

[0014] The temperature for the cell wall disruption treatment includes 4.0–6.0℃; and / or,

[0015] The pressure for the cell wall breaking process includes 800–1000 MPa.

[0016] In one embodiment, in step S20:

[0017] The amount of the compound enzyme added is 1%-2% of the mass of the *Micrococcus pseudocarpa* sample; wherein the cellulase activity is 50 U / mg and the alkaline protease activity is 200 U / mg. And / or,

[0018] In the complex enzyme, the mass ratio of the cellulase to the alkaline protease is 1:(2-4), wherein the cellulase activity is 500 U / mg and the alkaline protease activity is 200 U / mg; and / or,

[0019] The hydrolysis temperature is 50–60°C; and / or,

[0020] The hydrolysis time is 6–12 hours; and / or,

[0021] The centrifugation conditions include a centrifugation speed of 4000–6000 r / min; and / or,

[0022] The enzyme inactivation step includes heating at 80-90℃ for 15-20 minutes.

[0023] In one embodiment, in step S30: the salting-out reaction includes: mixing the supernatant with the ammonium sulfate to carry out the salting-out reaction, thereby obtaining the microalgae protein.

[0024] In one embodiment, step S30 involves post-processing the precipitated *Micrococcus pseudocarpa* protein, which includes: centrifuging the precipitated *Micrococcus pseudocarpa* protein, discarding the supernatant to obtain the *Micrococcus pseudocarpa* protein, mixing the *Micrococcus pseudocarpa* protein with water to obtain a mixture, and spray-drying the mixture to obtain *Micrococcus pseudocarpa* protein with high ACE inhibitory activity.

[0025] In one embodiment, the mass ratio of the microalgal protein to the water is 1:19-20.

[0026] The present invention also proposes a micrococcus protein, which is prepared by the above-mentioned method for preparing a micrococcus protein with high ACE inhibitory activity.

[0027] In the technical solution provided by this invention, the cell wall disruption step effectively destroys the cell wall structure of *Chlorella pseudocarpa*. Then, the addition of a mixed enzyme of cellulase and alkaline protease further degrades the cell wall and other components that hinder protein release, allowing more protein to be released into the solution and improving extraction efficiency. The enzyme treatment method helps maintain the natural structure and functional properties of the protein, avoiding denaturation or inactivation caused by harsh conditions, thus ensuring protein quality and bioavailability. Finally, the obtained *Chlorella pseudocarpa* protein is mostly of low molecular weight and has high ACE inhibitory activity. Furthermore, the process of this invention is simple to operate, uses *Chlorella pseudocarpa* powder or wet algae as raw material, has low production costs, and is pollution-free, significantly reducing the production cost of *Chlorella pseudocarpa* protein. No toxic substances are used throughout the process, making its application in the food processing field safer. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The molecular weight distribution of the microalgal protein with high ACE inhibitory activity prepared in Example 1 of this invention is shown in the figure.

[0030] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them.

[0032] It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination should be considered non-existent and not within the scope of protection claimed by this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0033] Currently, proteins are prepared from *Micrococcus pseudochlorella* using freeze-thaw salt precipitation, enzymatic extraction-assisted methods, and alkali-soluble acid precipitation methods. However, the prepared proteins have the following shortcomings: First, the defatted *Micrococcus pseudochlorella* proteins have poor water solubility and lose functional properties such as emulsification and foaming properties. Second, the dissolved proteins are not effectively extracted. Third, the preparation methods are relatively complex and the production cost is high.

[0034] In view of this, the present invention proposes a method for preparing a microphyte protein with high ACE inhibitory activity, comprising the following steps:

[0035] S10. Provide a sample of *Micrococcus pseudocarpa*, mix the sample with a buffer solution, adjust the pH value to 8-10, and then perform cell wall disruption treatment to obtain a mixed solution.

[0036] S20. The mixture is mixed with the complex enzyme, hydrolyzed, enzyme inactivated, and centrifuged to obtain a supernatant, wherein the complex enzyme includes cellulase and alkaline protease.

[0037] S30. The supernatant is subjected to a salting-out reaction to precipitate *Chlorella pseudocaryophyllum* protein. The precipitated *Chlorella pseudocaryophyllum* protein is then post-processed to obtain *Chlorella pseudocaryophyllum* protein with high ACE inhibitory activity. In the technical solution provided by this invention, the initial cell wall disruption step effectively destroys the cell wall structure of *Chlorella pseudocaryophyllum*. The subsequent addition of a mixed enzyme of cellulase and alkaline protease further degrades the cell wall and other components that hinder protein release, allowing more protein to be released into the solution and improving extraction efficiency. The enzyme treatment method helps maintain the natural structure and functional properties of the protein, avoiding protein denaturation or inactivation due to harsh conditions, thereby ensuring protein quality and bioavailability. Furthermore, this invention features a simple process, uses *Chlorella pseudocaryophyllum* samples as raw materials, has low production costs, and produces no pollution, significantly reducing the production cost of *Chlorella pseudocaryophyllum* protein. The entire process does not use toxic substances, making its application in the food processing field safer.

[0038] It's important to note that high ACE inhibitory activity means a substance possesses a strong ability to inhibit angiotensin-converting enzyme (ACE). ACE is an important enzyme that plays a crucial role in the body's blood pressure regulation mechanism. Specifically, ACE converts angiotensin I into angiotensin II, a potent vasoconstrictor. It also increases aldosterone secretion, leading to sodium and water retention and consequently elevated blood pressure. Therefore, substances with high ACE inhibitory activity can reduce the production of angiotensin II by inhibiting this conversion process, thereby helping to dilate blood vessels, lower blood pressure, and reduce the burden on the heart.

[0039] In some embodiments, in step S10, the *Nannochloropsis gaditana* sample includes *Nannochloropsis gaditana* powder or wet algae. That is, the *Nannochloropsis gaditana* sample can be either *Nannochloropsis gaditana* powder or wet algae. Specifically, the *Nannochloropsis gaditana* sample is sourced from the freshwater algae strain bank of the National Aquatic Biological Germplasm Bank, *Nannochloropsis gaditana* FACHB-926, and is obtained by culturing and collecting algae powder obtained by spray drying or by directly centrifuging and discarding the supernatant.

[0040] It should be noted that adjusting the pH value to a slightly alkaline environment of 8-10 is more conducive to the catalytic activity of alkaline protease and makes it easier to break the cell wall.

[0041] In some embodiments, in step S10:

[0042] The buffer solution includes phosphate buffer. Using phosphate buffer solution is beneficial for the stable existence of proteins and the expression of enzyme activity. The addition ratio of the *Micrococcus pseudocarpa* sample to the buffer solution is: 15-20 mL of phosphate buffer solution for every 1 g of *Micrococcus pseudocarpa* sample. It should be noted that when the *Micrococcus pseudocarpa* sample is wet algae, the corresponding amount is calculated based on dry weight, that is, 15-20 mL of phosphate buffer solution is added for every 1 g of wet algae (dry weight). Within the above range, the material can be fully dispersed while improving the efficiency of cell wall disruption and enzymatic hydrolysis.

[0043] In some embodiments, in step S10, the conditions for the cell wall disruption treatment include:

[0044] The temperature range for the cell disruption treatment is 4.0–6.0°C. Within this temperature range, it is beneficial for the cell structure to open up and release intracellular proteins, while also avoiding protein denaturation caused by high temperatures.

[0045] The pressure for the cell disruption treatment includes 800–1000 MPa. Within this pressure range,

[0046] In some embodiments, in step S20: the amount of the compound enzyme added is 1%-2% of the mass of the *Micrococcus pseudocarpa* sample; cellulase is used to break down the cellulose structure of the cell wall, which helps to release intracellular proteins, while alkaline protease can decompose large molecular proteins in algal powder to generate small molecular peptides with ACE inhibitory activity. The synergistic effect of the two enzymes can improve the protein recovery rate and the yield of functional peptides. Within the above-mentioned addition range, functional peptides can be hydrolyzed to the maximum extent.

[0047] In some embodiments, in the composite enzyme, the mass ratio of the cellulase to the alkaline protease is 1:(2-4), wherein the cellulase has an enzyme activity of 500 U / mg and the alkaline protease has an enzyme activity of 200 U / mg. Within this range, the protein extraction efficiency can be further improved.

[0048] In some embodiments, the hydrolysis temperature is 50–60°C; the hydrolysis time is 6–12 hours. Within this range, the hydrolysis can be ensured to be complete and efficient.

[0049] In some embodiments, the centrifugation conditions include centrifugation at 4000-6000 r / min for 15-30 min, which can separate undissolved matter and enzyme residue to obtain a relatively pure functional protein supernatant.

[0050] In some embodiments, the enzyme inactivation step includes heating at 80-90°C for 15-20 minutes. This step can briefly heat and inactivate residual enzymes to avoid unnecessary subsequent reactions.

[0051] In some embodiments, in step S30: the salting-out reaction includes: mixing the supernatant with the ammonium sulfate to carry out a salting-out reaction to obtain microalgae protein, and using ammonium sulfate to separate the protein by salting out, thus preserving its biological activity.

[0052] In some embodiments, step S30 includes the following steps: after centrifuging the precipitated *Micrococcus pseudocarpa* protein, discarding the supernatant to obtain *Micrococcus pseudocarpa* protein, mixing the *Micrococcus pseudocarpa* protein with water to obtain a mixture, and spray-drying the mixture to obtain *Micrococcus pseudocarpa* protein with high ACE inhibitory activity.

[0053] To ensure the purity of the protein obtained by salting out, a post-processing step can be performed: the precipitated *Chlorella pseudocaryophyllum* protein is redissolved in drinking water or other suitable buffer solution. This step is to remove residual ammonium sulfate. The aqueous solution with the redissolved protein is then spray-dried to remove moisture and obtain dried *Chlorella pseudocaryophyllum* protein powder, thus preserving the active ingredients of the substance.

[0054] In some embodiments, the mass ratio of the microchlorella protein to the water is 1:19 to 20. Within this range, the microchlorella protein is fully dissolved, which also facilitates subsequent spray drying and preserves the activity of the substance.

[0055] This invention also proposes a *Micrococcus pseudocarpa* protein, which is prepared by the above-described method for preparing *Micrococcus pseudocarpa* protein with high ACE inhibitory activity. The *Micrococcus pseudocarpa* protein possesses all the technical features of the above-described method for preparing *Micrococcus pseudocarpa* protein with high ACE inhibitory activity, and therefore has the aforementioned technical effects, which will not be elaborated upon further here.

[0056] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0057] Experimental materials

[0058] The *Nannochloropsisgaditana* sample was obtained from the freshwater algae strain bank of the National Aquatic Organism Germplasm Resource Bank, specifically from *Nannochloropsisgaditana* FACHB-926, which was cultured, collected, and then spray-dried to obtain algal powder.

[0059] Example 1

[0060] This embodiment provides a micrococcus-like protein with high ACE inhibitory activity. The preparation method of the micrococcus-like protein with high ACE inhibitory activity includes the following steps:

[0061] S10. Mix 10g of *Micrococcus pseudocarpa* powder with 200ml of phosphate buffer, adjust the pH of the mixed solution to 9, and perform cell wall disruption treatment at 5.0℃ and 1000Mpa to obtain a mixed solution.

[0062] S20. The mixture is mixed with the compound enzyme, hydrolyzed at 55°C for 8 hours, heated at 80°C for 20 minutes to inactivate the enzyme, and then centrifuged at 5000 r / min for 20 minutes to obtain the supernatant. The compound enzyme includes cellulase and alkaline protease, and the mass ratio of cellulase to alkaline protease is 1:4. The cellulase activity is 500 U / mg and the alkaline protease activity is 200 U / mg.

[0063] S30. Take the supernatant from step S20 and mix it with ammonium sulfate at an added amount of 0.261 g / mL to carry out a salting-out reaction to precipitate Micrococcus pseudomicrophylla protein. The precipitated Micrococcus pseudomicrophylla protein is then post-processed by centrifuging it and mixing it with water at a ratio of 1:10 to obtain Micrococcus pseudomicrophylla protein with high ACE inhibitory activity.

[0064] Example 2

[0065] This embodiment provides a micrococcus-like protein with high ACE inhibitory activity. The preparation method of the micrococcus-like protein with high ACE inhibitory activity includes the following steps:

[0066] S10. Mix 10g of *Micrococcus pseudocarpa* powder with 200ml of phosphate buffer, adjust the pH of the mixed solution to 10, and perform cell wall disruption treatment at 5.0℃ and 1000Mpa to obtain a mixed solution.

[0067] S20. The mixture is mixed with the compound enzyme, hydrolyzed at 55°C for 8 hours, heated at 80°C for 20 minutes to inactivate the enzyme, and then centrifuged at 5000 r / min for 20 minutes to obtain the supernatant. The compound enzyme includes cellulase and alkaline protease, and the mass ratio of cellulase to alkaline protease is 1:4.

[0068] S30. Take the supernatant from step S20 and mix it with ammonium sulfate at an added amount of 0.261 g / mL to carry out a salting-out reaction to precipitate Micrococcus pseudomicrophylla protein. The precipitated Micrococcus pseudomicrophylla protein is then post-processed by centrifuging it and mixing it with water at a ratio of 1:10 to obtain Micrococcus pseudomicrophylla protein with high ACE inhibitory activity.

[0069] Example 3

[0070] This embodiment provides a micrococcus-like protein with high ACE inhibitory activity. The preparation method of the micrococcus-like protein with high ACE inhibitory activity includes the following steps:

[0071] S10. Mix 10g of *Micrococcus pseudocarpa* powder with 200ml of phosphate buffer, adjust the pH of the mixed solution to 7, and perform cell wall disruption treatment at 5.0℃ and 1000Mpa to obtain a mixed solution.

[0072] S20. The mixture is mixed with the compound enzyme, hydrolyzed at 40°C for 4 hours, heated at 80°C for 20 minutes to inactivate the enzyme, and then centrifuged at 5000 r / min for 20 minutes to obtain the supernatant. The compound enzyme includes cellulase and alkaline protease, and the mass ratio of cellulase to alkaline protease is 1:4.

[0073] S30. Take the supernatant from step S20 and mix it with ammonium sulfate at an added amount of 0.261 g / mL to carry out a salting-out reaction to precipitate Micrococcus pseudomicrophylla protein. The precipitated Micrococcus pseudomicrophylla protein is then post-processed by centrifuging it and mixing it with water at a ratio of 1:10 to obtain Micrococcus pseudomicrophylla protein with high ACE inhibitory activity.

[0074] Example 4

[0075] This embodiment provides a micrococcus-like protein with high ACE inhibitory activity. The preparation method of the micrococcus-like protein with high ACE inhibitory activity includes the following steps:

[0076] S10. Mix 10g of *Micrococcus pseudocarpa* powder with 200ml of phosphate buffer, adjust the pH of the mixed solution to 5, and perform cell wall disruption treatment at 5.0℃ and 1000Mpa to obtain a mixed solution.

[0077] S20. The mixture is mixed with the compound enzyme, hydrolyzed at 55°C for 8 hours, heated at 80°C for 20 minutes to inactivate the enzyme, and then centrifuged at 5000 r / min for 20 minutes to obtain the supernatant. The compound enzyme includes cellulase and alkaline protease, and the mass ratio of cellulase to alkaline protease is 1:4.

[0078] S30. Take the supernatant from step S20 and mix it with ammonium sulfate at an added amount of 0.261 g / mL to carry out a salting-out reaction to precipitate Micrococcus pseudomicrophylla protein. The precipitated Micrococcus pseudomicrophylla protein is then post-processed by centrifuging it and mixing it with water at a ratio of 1:10 to obtain Micrococcus pseudomicrophylla protein with high ACE inhibitory activity.

[0079] Example 5

[0080] This embodiment provides a micrococcus-like protein with high ACE inhibitory activity. The preparation method of the micrococcus-like protein with high ACE inhibitory activity includes the following steps:

[0081] S10. Mix 10g of *Micrococcus pseudocarpa* powder with 200ml of phosphate buffer, adjust the pH of the mixed solution to 7, and perform cell wall disruption treatment at 5.0℃ and 1000Mpa to obtain a mixed solution.

[0082] S20. The mixture is mixed with the compound enzyme, hydrolyzed at 55°C for 8 hours, heated at 80°C for 20 minutes to inactivate the enzyme, and then centrifuged at 5000 r / min for 20 minutes to obtain the supernatant. The compound enzyme includes cellulase and alkaline protease, and the mass ratio of cellulase to alkaline protease is 1:2.

[0083] S30. Take the supernatant from step S20 and mix it with ammonium sulfate at an added amount of 0.261 g / mL to carry out a salting-out reaction to precipitate Micrococcus pseudomicrophylla protein. The precipitated Micrococcus pseudomicrophylla protein is then post-processed by centrifuging it and mixing it with water at a ratio of 1:10 to obtain Micrococcus pseudomicrophylla protein with high ACE inhibitory activity.

[0084] Example 6

[0085] This embodiment provides a micrococcus-like protein with high ACE inhibitory activity. The preparation method of the micrococcus-like protein with high ACE inhibitory activity includes the following steps:

[0086] S10. Mix 10g of *Micrococcus pseudocarpa* powder with 200ml of phosphate buffer, adjust the pH of the mixed solution to 6, and perform cell wall disruption treatment at 5.0℃ and 1000Mpa to obtain a mixed solution.

[0087] S20. The mixture is mixed with cellulase, hydrolyzed at 55°C for 4 hours, heated at 80°C for 20 minutes to inactivate the enzyme, cooled, and the pH is adjusted to 9. Alkaline protease is added, hydrolyzed at 55°C for 4 hours, heated at 80°C for 20 minutes to inactivate the enzyme, and then centrifuged at 5000 r / min for 20 minutes to obtain the supernatant. The complex enzyme includes cellulase and alkaline protease, and the mass ratio of cellulase to alkaline protease is 1:4.

[0088] S30. Take the supernatant from step S20 and mix it with ammonium sulfate at an added amount of 0.261 g / mL to carry out a salting-out reaction to precipitate Micrococcus pseudomicrophylla protein. The precipitated Micrococcus pseudomicrophylla protein is then post-processed by centrifuging it and mixing it with water at a ratio of 1:10 to obtain Micrococcus pseudomicrophylla protein with high ACE inhibitory activity.

[0089] Comparative Example 1

[0090] Except for removing "perform cell wall disruption treatment at 5.0℃ and 1000Mpa" in step S10, the rest is the same as in Example 1.

[0091] Comparative Example 2

[0092] Except for the fact that the complex enzyme in step S20 is only alkaline protease, the rest is the same as in Example 1.

[0093] Comparative Example 3

[0094] Except for the fact that the complex enzyme in step S20 is only cellulase, everything else is the same as in Example 1.

[0095] Comparative Example 4

[0096] Except for replacing the complex enzyme in step S20 with papain and pectinase, the rest is the same as in Example 1.

[0097] Comparative Example 5

[0098] Except for the fact that the complex enzyme in step S20 includes cellulase and alkaline protease, and the mass ratio of the cellulase and the alkaline protease is 1:1, the rest is the same as in Example 1.

[0099] Comparative Example 6

[0100] Except for replacing the complex enzyme in step S20 with papain and pectinase, and the difference that the mass ratio of papain and pectinase is 1:1, everything else is the same as in Example 1.

[0101] Performance testing

[0102] The yield and ACE inhibitory activity of *Micrococcus pseudocarpa* proteins with high ACE inhibitory activity obtained in Examples 1-6 and Comparative Examples 1-6 were tested using a steady-state method. The determination of the *Micrococcus pseudocarpa* protein yield was as follows:

[0103] The protein mass in *Micrococcus pseudochlorella* was determined by the Kjeldahl method, and then converted to an equivalent mass of algal powder.

[0104] 2. Assay of ACE inhibitory activity of Micrococcus pseudocarpa protein:

[0105]

[0106] The measurement method was ultraviolet spectrophotometry, ΔA 空白 The ACE inhibition rate of 5 mmol / L captopril; ΔA 测定 The ACE inhibition rate of the sample is shown in Table 1.

[0107] Table 1

[0108] Test items Yield (%) ACE inhibitory activity (%) Remark Example 1 83.14 68.16 Broken wall Example 2 63.14 59.51 Mixed enzymes (pH=10) Example 3 79.11 63.67 Mixed enzymes (pH=7) Example 4 66.72 60.37 Mixed enzymes (pH=5) Example 5 75.34 63.67 Enzyme ratio (1:4, pH=7) Example 6 68.91 64.16 Add cellulase first, then add alkaline protease. Comparative Example 1 61.48 62.02 Unprocessed Comparative Example 2 67.61 65.33 alkaline protease Comparative Example 3 58.52 30.11 Cellulase Comparative Example 4 65.98 34.54 Different mixed enzymes Comparative Example 5 70.86 43.83 Enzyme ratio (1:1, pH=7) Comparative Example 6 57.45 35.82 Add pectinase first, then add papain.

[0109] As shown in Table 1, compared with the comparative examples under different conditions in Examples 1-5, the yield and ACE inhibitory activity of *Chlorella pseudocaryophyllum* protein were all improved. Furthermore, comparison of the experimental groups in different examples also revealed that the protein extraction rate of *Chlorella pseudocaryophyllum* varied under different conditions, which is related to the optimal reaction conditions of the mixed enzyme. Clearly, the method of the present invention can significantly improve the yield and ACE inhibitory activity of *Chlorella pseudocaryophyllum* protein, making the extraction of *Chlorella pseudocaryophyllum* protein simpler and more efficient.

[0110] Simultaneously, the molecular weight of *Microcystis aeruginosa* protein was determined by high-performance gel filtration chromatography (GPC) in this invention, and the molecular weight test results of the *Microcystis aeruginosa* protein prepared by this method are as follows: Figure 1 As shown, the molecular weight of *Micrococcus pseudocarpa* proteins ranges from 0 to 10 kDa, exhibiting small molecular weights. Specifically, 0-1 kDa proteins account for 50.95%, 1-3 kDa proteins for 37.15%, and 3-10 kDa proteins for 11.90%. It should be noted that small-molecule proteins possess stronger ACE inhibitory activity. Small-molecule proteins or peptides experience less steric hindrance when interacting with ACE, allowing them to more freely approach the active site of ACE and effectively interact with the amino acid residues at the active site. In contrast, large-molecule proteins, due to their complex structure and large molecular weight, may experience steric hindrance when approaching the ACE active site, affecting their binding efficiency and thus reducing ACE inhibitory activity.

[0111] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A method for preparing a microphyte-like protein with high ACE inhibitory activity, characterized in that, Includes the following steps: S10. Provide a sample of *Micrococcus pseudocarpa*, mix the sample with a buffer solution, adjust the pH value to 8-10, and then perform cell wall disruption treatment to obtain a mixed solution. S20. The mixture is mixed with the complex enzyme, enzymatically hydrolyzed, enzyme inactivated, and centrifuged to obtain a supernatant, wherein the complex enzyme includes cellulase and alkaline protease. S30. The supernatant is subjected to a salting-out reaction to precipitate Micrococcus pseudocarpa protein. The precipitated Micrococcus pseudocarpa protein is then post-processed to obtain Micrococcus pseudocarpa protein with high ACE inhibitory activity.

2. The method for preparing the microalgae protein with high ACE inhibitory activity as described in claim 1, characterized in that, In step S10, the *Micrococcus pseudocarpa* sample includes *Micrococcus pseudocarpa* powder or wet algae.

3. The method for preparing the microalgae protein with high ACE inhibitory activity as described in claim 2, characterized in that, In step S10: The buffer solution includes phosphate buffer, and the addition ratio of the *Micrococcus pseudocarpa* powder or wet algae to the buffer solution is: 15-20 mL of phosphate buffer is added for every 1 g of *Micrococcus pseudocarpa* sample.

4. The method for preparing the microalgae protein with high ACE inhibitory activity as described in claim 3, characterized in that, In step S10, the conditions for the cell wall disruption process are as follows: The temperature for the cell wall disruption treatment includes 4.0–6.0℃; and / or, The pressure for the cell wall breaking process includes 800–1000 MPa.

5. The method for preparing the microalgae protein with high ACE inhibitory activity as described in claim 1, characterized in that, In step S20: The amount of the compound enzyme added is 1%-2% of the mass of the *Micrococcus pseudocarpa* sample; and / or, In the complex enzyme, the mass ratio of the cellulase to the alkaline protease is 1:(2-4), wherein the cellulase activity is 500 U / mg and the alkaline protease activity is 200 U / mg; and / or, The hydrolysis temperature is 50–60°C; and / or, The hydrolysis time is 6–12 hours; and / or, The centrifugation conditions include centrifugation at 4000–6000 r / min for 15–30 min; and / or, The enzyme inactivation step includes heating at 80-90℃ for 15-20 minutes.

6. The method for preparing the microalgae protein with high ACE inhibitory activity as described in claim 1, characterized in that, In step S30: the salting-out reaction includes: mixing the supernatant with the ammonium sulfate to carry out the salting-out reaction, thereby obtaining the microalgae protein.

7. The method for preparing the microalgae protein with high ACE inhibitory activity as described in claim 1, characterized in that, In step S30: the post-processing step of the precipitated *Micrococcus pseudocarpa* protein includes: centrifuging the precipitated *Micrococcus pseudocarpa* protein, discarding the supernatant to obtain *Micrococcus pseudocarpa* protein, mixing the *Micrococcus pseudocarpa* protein with water to obtain a mixture, and spray drying the mixture to obtain *Micrococcus pseudocarpa* protein with high ACE inhibitory activity.

8. The method for preparing the microalgae protein with high ACE inhibitory activity as described in claim 7, characterized in that, The mass ratio of the microalgal protein to the water is 1:19-20.

9. A microalgae-like protein, characterized in that, The microchloride protein is prepared by the method for preparing microchloride protein with high ACE inhibitory activity as described in any one of claims 1 to 8.

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