A method for preparing a superoxide dismutase from saccharomyces cerevisiae

Abstract

The present application relates to the technical field of bioactive enzyme preparation, and discloses a preparation method of superoxide dismutase from saccharomyces cerevisiae. The method comprises the following steps: inoculating saccharomyces cerevisiae to obtain a fermentation liquor; centrifuging to collect bacterial bodies, and then washing and resuspending the bacterial bodies by using a buffer to obtain a bacterial body suspension; performing freeze-thaw treatment and glass bead oscillation wall-breaking treatment on the bacterial body suspension to obtain a broken wall liquid; performing low-temperature centrifugal separation on the broken wall liquid, and collecting supernatant to obtain a crude enzyme liquid; performing two-stage ammonium sulfate salting-out and dialysis desalting on the crude enzyme liquid to obtain an SOD active component; and adding glycine, trehalose and mannitol into the SOD active component to perform stabilization treatment, so as to obtain an SOD product. The present application does not need to rely on high-pressure homogenization, ultrasonic wall-breaking and chromatography purification equipment, the process condition is mild, and the present application is suitable for the preparation of the SOD product from saccharomyces cerevisiae.

Description

Technical Field

[0001] This invention relates to the field of bioactive enzyme preparation technology, specifically a method for preparing superoxide dismutase derived from Saccharomyces cerevisiae. Background Technology

[0002] Superoxide dismutase (SOD) is a class of metallo-oxidizing enzymes widely found in animals, plants, and microorganisms. It specifically catalyzes the dismutation reaction of superoxide anion free radicals, generating oxygen and hydrogen peroxide, thereby effectively scavenging excess reactive oxygen species in organisms and reducing oxidative stress damage. Due to its important roles in antioxidation, anti-aging, anti-inflammation, and enhancing the body's defense capabilities, SOD has received widespread attention in the food, health product, cosmetic, and biopharmaceutical fields in recent years. Compared to SOD derived from animal and plant tissues, SOD derived from microorganisms has advantages such as shorter culture cycles, stable raw material sources, lower production costs, and ease of large-scale preparation. Among them, *Saccharomyces cerevisiae*, as a recognized safe industrial microorganism with mature culture conditions, has good application potential as a strain for SOD production.

[0003] Currently, the extraction and preparation of yeast-derived SOD mostly employs high-pressure homogenization or ultrasonic cell disruption to break down cells, followed by ultrafiltration, chromatographic purification, or freeze-drying to obtain the target enzyme product. However, these processes typically suffer from drawbacks such as high equipment investment, high energy consumption, complex operation, and stringent process conditions. In particular, high-pressure homogenization and ultrasonic treatment are prone to localized overheating, leading to partial loss of SOD activity. Furthermore, while subsequent chromatographic purification can improve purity, the equipment is expensive and time-consuming, making it unsuitable for low-cost, large-scale production. In addition, existing processes do not adequately address the long-term storage stability of SOD products, lacking effective stabilization methods, resulting in a rapid decline in enzyme activity during storage. Therefore, developing a method for preparing SOD from Saccharomyces cerevisiae that does not rely on high-pressure homogenization, ultrasonication, and chromatographic equipment, while simultaneously ensuring efficient cell disruption, maintaining enzyme activity, and ensuring storage stability, has significant practical application value. Summary of the Invention

[0004] To address the problems mentioned in the background section, the present invention provides the following technical solution: a method for preparing superoxide dismutase from *Saccharomyces cerevisiae*, comprising the following steps:

[0005] (1) Inoculate the brewing yeast into the culture medium and culture it to obtain the fermentation broth;

[0006] (2) Centrifuge the fermentation broth, collect the cells, and wash and resuspend the cells with buffer solution to obtain a cell suspension;

[0007] (3) The bacterial suspension is subjected to freeze-thaw treatment and glass bead shaking to break the cell wall, so as to obtain a cell wall broken liquid; the freeze-thaw treatment is performed 1 to 4 times, the freezing temperature is -20 to -40℃, and the thawing temperature is 20 to 30℃; the glass bead particle size is 0.3 to 0.8 mm, the mass-volume ratio of glass beads to bacterial suspension is 0.2:1 to 1.0:1, and the shaking time is 10 to 40 min;

[0008] (4) The cell wall-breaking liquid is subjected to low-temperature centrifugation to separate the supernatant and obtain crude enzyme solution;

[0009] (5) The crude enzyme solution is subjected to two stages of ammonium sulfate precipitation and dialysis desalting to obtain SOD active components; in the two stages of ammonium sulfate precipitation, the first stage is 35% to 40% saturation and the second stage is 70% to 80% saturation.

[0010] (6) Add glycine, trehalose and mannitol to the SOD active component for stabilization treatment, adjust the pH to 7.0 to 7.5, and let it stand at 4°C for 2 to 12 hours to obtain the SOD product; the amount of glycine added is 0.3% to 0.6%, the amount of trehalose added is 2.0% to 4.0%, and the amount of mannitol added is 1.0% to 2.0%.

[0011] Preferably, the freeze-thaw treatment in step (3) is performed 3 times, the glass beads have a particle size of 0.5 mm, the mass-to-volume ratio of glass beads to bacterial suspension is 0.5:1, and the shaking time is 20 min.

[0012] Preferably, the glass bead oscillation and cell wall breaking treatment in step (3) is carried out in an ice bath or at 4°C.

[0013] Preferably, the low-temperature centrifugation separation conditions in step (4) are: centrifugation temperature 2-8℃, centrifugation speed 8000-12000r / min, centrifugation time 10-25min; after centrifugation, further filtration and clarification are carried out using a 0.45 μm microporous membrane.

[0014] Preferably, the salt-precipitate obtained in step (5) is reconstituted using Tris-HCl buffer or phosphate buffer, wherein the pH of the reconstitution buffer is 7.0 to 8.0; the dialysis desalination in step (5) uses a dialysis bag with a molecular weight cutoff of 8kDa to 14kDa, and is dialyzed at 4°C for 8 to 24 hours, with the solution changed 2 to 4 times during the dialysis process.

[0015] Preferably, in step (6), the amount of glycine added is 0.5%, the amount of trehalose added is 3.0%, the amount of mannitol added is 1.5%, and after adjusting the pH to 7.2, it is allowed to stand at 4°C for 4 hours to equilibrate.

[0016] Preferably, in step (6), the amount of glycine added is 0.3%, the amount of trehalose added is 4.0%, the amount of mannitol added is 2.0%, and after adjusting the pH to 7.2, it is allowed to stand at 4°C for 4 hours to equilibrate.

[0017] Preferably, the SOD product is a liquid or solid formulation; when it is a liquid formulation, it should be stored at 2–8°C in the dark.

[0018] Preferably, after the SOD product is diluted or reconstituted according to the testing requirements, the SOD enzyme activity is 900-1100 U / mL.

[0019] Preferably, the enzyme activity retention rate of the SOD product is 84% ​​to 91% after being stored at 4°C for 6 months.

[0020] Compared with the prior art, the present invention provides a method for preparing superoxide dismutase from Saccharomyces cerevisiae, which has the following beneficial effects:

[0021] 1. This invention uses brewer's yeast as raw material, which has a short strain culture cycle, stable raw material source and high safety, making it easy to scale up industrial production; at the same time, it does not require the use of genetically engineered bacteria or special induction conditions, resulting in low production costs and easy promotion and application.

[0022] 2. This invention uses a combination of freeze-thaw treatment and glass bead oscillation to disrupt cell walls, replacing traditional high-pressure homogenization or ultrasonic disruption. The freeze-thaw process loosens the cell membrane structure and increases its permeability. Combined with the mechanical shearing effect of the glass beads, this can improve the release efficiency of intracellular SOD in Saccharomyces cerevisiae. Moreover, the entire process can be carried out in an ice bath or at 4°C, which helps to reduce the impact of high temperature and high pressure on SOD activity. The SOD enzyme activity recovery rate after disruption can reach more than 60%.

[0023] 3. This invention utilizes a two-stage ammonium sulfate salting-out and dialysis desalting fractionation system to achieve the enrichment and desalting of SOD active components without relying on chromatography equipment. The system is simple to operate and low in cost. By controlling the first stage to remove impurities at 35%–40% saturation and the second stage to precipitate the target SOD at 70%–80% saturation, the specific activity of the purified SOD is significantly improved, making it suitable for large-scale industrial preparation.

[0024] 4. This invention uses glycine, trehalose, and mannitol in a specific ratio as a composite stabilizer for SOD. Utilizing the synergistic effect of amino acids, sugars, and sugar alcohols as protective agents, it protects SOD activity through multiple pathways, including maintaining the higher-order structure of the enzyme protein, reducing aggregation and precipitation, and scavenging free radicals. Experiments show that the enzyme activity retention rate of the three-component composite stabilization system can reach 84%–91% after storage at 4°C for 6 months. Under the conditions of the examples, the three-component composite stabilization system has a higher enzyme activity retention rate than single-stabilizer or two-component stabilizer systems, which is beneficial for improving the long-term storage stability of SOD products.

[0025] 5. The entire preparation process of this invention does not require expensive equipment such as high-pressure homogenizers, ultrasonic disruptors, and chromatography purification systems. The process conditions are mild, the operation is simple, and the reproducibility is good. The enzyme activity of the obtained SOD product can reach 900-1100 U / mL. It can be used directly as a liquid preparation or further dried to make a solid preparation. It is suitable for further development and application in the fields of cosmetics, food, and related biological products. Attached Figure Description

[0026] Figure 1 This is a schematic flowchart of a method for preparing superoxide dismutase from brewer's yeast according to the present invention.

[0027] Figure 2 A schematic diagram illustrating the impact of different cell wall disruption methods on SOD release.

[0028] Figure 3 This is a schematic diagram showing the impact of different stabilizer systems on the long-term storage stability of SOD products. Detailed Implementation

[0029] The technical solutions of 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] refer to Figures 1-3 :

[0031] In the following examples, the *Saccharomyces cerevisiae* strain used was a commercially available common *Saccharomyces cerevisiae*, and all raw materials were conventional reagents in the art. SOD activity was determined according to GB / T 41906-2022 "Method for Determination of Superoxide Dismutase Activity". Total protein content was determined using the Coomassie Brilliant Blue G-250 method. Specific activity = enzyme activity / protein content. Enzyme activity recovery rate (%) = total enzyme activity of samples at each purification stage / total enzyme activity of crude enzyme solution × 100%. Enzyme activity retention rate (%) = enzyme activity of the sample after storage / enzyme activity of the initial sample × 100%. Each example and comparative example had three parallel samples. Results are expressed as mean ± standard deviation or range.

[0032] Example 1

[0033] This embodiment provides a method for preparing superoxide dismutase from Saccharomyces cerevisiae, including the following steps:

[0034] (1) Cell culture: Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 30℃ and 180r / min for 24h with shaking to obtain fermentation broth.

[0035] (2) Cell isolation: The above fermentation broth was centrifuged at 6000 r / min for 10 min at 4℃, and the cells were collected. The cells were washed twice with 0.05 mol / L phosphate buffer (pH 7.2), and then resuspended at a cell wet weight to buffer mass-volume ratio of 1:5 to obtain a cell suspension.

[0036] (3) Synergistic cell disruption: The bacterial suspension was frozen at -20℃ for 2 hours and then thawed at 25℃. The freeze-thaw cycle was repeated 3 times. Then, glass beads with a particle size of 0.5 mm were added at a mass-volume ratio of glass beads to bacterial suspension of 0.5:1. The mixture was shaken on a vortex shaker for 20 minutes (under ice bath conditions) to obtain the cell disruption solution.

[0037] (4) Removal of impurities: Centrifuge the cell wall disruption solution at 10,000 r / min for 15 min at 4℃ to remove cell debris and insoluble impurities, collect the supernatant to obtain crude enzyme solution.

[0038] (5) Graded salting out: Add solid ammonium sulfate to the crude enzyme solution to 35% saturation, stir for 30 min at 4℃ and let stand for 20 min, then centrifuge to remove the precipitate; continue to add ammonium sulfate to the supernatant to 75% saturation, let stand for 1 h at 4℃ and then centrifuge to collect the precipitate, and reconstitute with a small amount of Tris-HCl buffer (0.02 mol / L, pH 7.8) to obtain the salting out enrichment solution.

[0039] (6) Desalting treatment: The salting-out enriched solution was placed into a dialysis bag with a molecular weight cutoff of 8kDa to 14kDa and dialyzed at 4℃ for 12h, with the solution changed 3 times during the period, to obtain the SOD active component.

[0040] (7) Stabilization treatment: Glycine (0.5%), trehalose (3.0%) and mannitol (1.5%) were added to the obtained SOD active components, the pH was adjusted to 7.2, and the mixture was allowed to stand at 4°C for 4 hours to obtain SOD liquid preparation.

[0041] Efficacy testing: After dilution according to testing requirements, the SOD product obtained in this embodiment showed an SOD enzyme activity of 980–1100 U / mL, with an average of 1040.6 ± 52.3 U / mL, a specific activity of 552.4 ± 31.8 U / mg, and an enzyme activity recovery rate of 66.8 ± 3.4%. After storage at 4°C for 6 months, the enzyme activity retention rate was 84%–88%, indicating that the SOD product prepared by the method of this invention has high activity and good storage stability.

[0042] Example 2

[0043] The only difference between this embodiment and Embodiment 1 is the number of freeze-thaw cycles in step (3). In this embodiment, the freeze-thaw process is repeated twice, and the remaining steps are the same as in Embodiment 1.

[0044] Efficacy testing: After dilution according to testing requirements, the SOD enzyme activity of the obtained SOD product was 900–1020 U / mL, with an average of 958.7 ± 47.1 U / mL, and the specific activity was 491.3 ± 28.6 U / mg. The results indicate that reducing the number of freeze-thaw cycles slightly decreases the degree of cell wall disruption, but a high-activity SOD product can still be obtained.

[0045] Example 3

[0046] The only difference between this embodiment and Embodiment 1 is the glass bead oscillation time in step (3). In this embodiment, the glass bead oscillation time is 15 minutes, and the remaining steps are the same as in Embodiment 1.

[0047] Efficacy testing: After dilution according to testing requirements, the SOD enzyme activity of the obtained SOD product was 930–1050 U / mL, with an average of 987.6 ± 50.2 U / mL, and the specific activity was 478.5 ± 30.4 U / mg. This indicates that appropriately extending the shaking time is beneficial for more complete SOD release.

[0048] Example 4

[0049] The only difference between this embodiment and Embodiment 1 is the different saturation levels in step (5) during the graded salting out. In this embodiment, the ammonium sulfate saturation level is 40% in the first stage and 80% in the second stage, with the remaining steps being the same as in Embodiment 1.

[0050] Efficacy testing: After dilution according to the testing requirements, the SOD enzyme activity of the obtained SOD product was 950–1080 U / mL, with an average of 1012.3 ± 48.6 U / mL, and the specific activity was 580.7 ± 35.2 U / mg. This indicates that appropriately increasing the salting-out saturation is beneficial for SOD enrichment, but care should be taken to avoid co-precipitation loss of the target enzyme.

[0051] Example 5

[0052] The only difference between this embodiment and Example 1 is the amount of stabilizer added in step (7). In this embodiment, 0.3% glycine, 4.0% trehalose, and 2.0% mannitol are added, and the remaining steps are the same as in Example 1.

[0053] Efficacy testing: After dilution according to testing requirements, the initial SOD enzyme activity of the obtained SOD product was 970–1090 U / mL, with an average of 1018.5 ± 49.3 U / mL, and a specific activity of 560.2 ± 32.5 U / mg. After storage at 4°C for 6 months, the enzyme activity retention rate was 88%–91%, higher than that of Example 1. This indicates that by optimizing the stabilizer ratio, the long-term storage stability of the SOD product can be further improved.

[0054] Comparative Example 1 (no synergistic cell disruption, only simple stirring)

[0055] The difference between this comparative example and Example 1 is that: in step (3), freeze-thaw treatment and glass bead oscillation to break the cell wall are not performed; the bacterial suspension is simply stirred and then centrifuged directly. The remaining steps are the same as in Example 1.

[0056] Efficacy testing: After dilution according to the testing requirements, the SOD enzyme activity of the obtained SOD product was only 420-600 U / mL, with an average of 510.3±45.2 U / mL, and the enzyme activity recovery rate was 31.6±3.2%. This indicates that without synergistic cell disruption treatment, the SOD release efficiency is extremely low, and effective extraction cannot be achieved.

[0057] Comparative Example 2 (Freeze-thaw treatment only)

[0058] The difference between this comparative example and Example 1 is that in step (3), the bacterial suspension is subjected to freeze-thaw treatment three times, glass beads are not added, and no shaking is performed to break the cell walls. The remaining steps are the same as in Example 1.

[0059] Effect test: After dilution according to the test requirements, the SOD enzyme activity of the obtained SOD product was 680-760 U / mL, with an average of 712.5±36.8 U / mL, and the enzyme activity recovery rate was 45.7±2.9%. This indicates that although the freeze-thaw treatment alone has a certain cell wall disruption effect, it is far lower than that of the synergistic cell wall disruption group.

[0060] Comparative Example 3 (Individual glass bead oscillation treatment)

[0061] The difference between this comparative example and Example 1 is that: in step (3), no freeze-thaw treatment is performed, and only 0.5 mm glass beads are added and shaken for 20 min. The remaining steps are the same as in Example 1.

[0062] Efficacy testing: After dilution according to testing requirements, the SOD enzyme activity of the obtained SOD product was 770–850 U / mL, with an average of 812.3 ± 39.7 U / mL, and the enzyme activity recovery rate was 52.4 ± 3.1%. This indicates that mechanical shaking alone is more effective than freeze-thaw alone in disrupting the cell wall, but still lower than the synergistic disruption group. The comparison results between Comparative Examples 1-3 and Example 1 are as follows: Figure 2 As shown.

[0063] Depend on Figure 2 It is evident that the freeze-thaw and glass bead oscillation synergistic cell disruption group showed higher SOD activity and enzyme activity recovery rates than the single treatment group, indicating that the two have a synergistic promoting effect.

[0064] Comparative Example 4 (without stabilizer)

[0065] The difference between this comparative example and Example 1 is that glycine, trehalose and mannitol are not added in step (7), while the other steps are the same as in Example 1.

[0066] Effect test: The initial enzyme activity of the obtained SOD product was 960-1080 U / mL, which was close to that of Example 1; however, after being stored at 4°C for 6 months, the enzyme activity retention rate was only 58%-68%, indicating that the SOD product suffered serious enzyme activity loss during long-term storage without stabilizers.

[0067] Comparative Example 5 (Comparison of single-component stabilizer and two-component stabilizer)

[0068] This comparative example included multiple controls to investigate the effects of single-component and two-component stabilizers on the storage stability of SOD products. The preparation processes for each control group were the same as in Example 1, except for the stabilizer system in step (7). The enzyme activity retention rates of each group after 6 months of storage at 4°C are shown below. Figure 3 As shown.

[0069] Depend on Figure 3 It is known that both single and two-component stabilizers can improve the storage stability of SOD to a certain extent, but the three-component composite stabilizer system of glycine, trehalose, and mannitol is more effective than the single or two-component system. Example 5, by optimizing the ratio of the three components, achieved an enzyme activity retention rate of 88%–91% after 6 months, demonstrating that the composite stabilizer selected in this invention has a synergistic protective effect.

[0070] Stability verification examples

[0071] The SOD products obtained in Examples 1, 5, and Comparative Example 4 were subjected to stability studies at 2–8°C and 25°C for 6 months, respectively, with monthly samples taken to measure residual enzyme activity. The results showed that the enzyme activity retention rates of Examples 1 and 5 were ≥84% at 2–8°C for 6 months, and approximately 72% and 76% at 25°C for 6 months, respectively; while the enzyme activity retention rate of Comparative Example 4 was significantly lower under the same conditions. This indicates that the stabilization treatment of the present invention can effectively improve the storage stability of SOD products at different temperatures.

[0072] Application examples

[0073] In one application, the SOD product can be added as an active enzyme ingredient to cosmetic formulations, such as skin lotions, serums, face masks, lotions, creams, or sprays.

[0074] In summary, the method for preparing superoxide dismutase (SOD) from Saccharomyces cerevisiae provided by this invention, through freeze-thaw and glass bead oscillation synergistic cell disruption, two-stage ammonium sulfate precipitation, and three-component composite stabilizer treatment, can obtain SOD products with good activity and storage stability without the need for high-pressure homogenization, ultrasonication, and chromatography equipment, and is suitable for the preparation of SOD products from Saccharomyces cerevisiae.

[0075] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing superoxide dismutase derived from Saccharomyces cerevisiae, characterized in that, Includes the following steps: (1) Inoculate the brewing yeast into the culture medium and culture it to obtain the fermentation broth; (2) Centrifuge the fermentation broth, collect the cells, and wash and resuspend the cells with buffer solution to obtain a cell suspension; (3) The bacterial suspension is subjected to freeze-thaw treatment and glass bead shaking to break the cell wall, so as to obtain a cell wall broken liquid; the freeze-thaw treatment is performed 1 to 4 times, the freezing temperature is -20 to -40℃, and the thawing temperature is 20 to 30℃; the glass bead particle size is 0.3 to 0.8 mm, the mass-volume ratio of glass beads to bacterial suspension is 0.2:1 to 1.0:1, and the shaking time is 10 to 40 min; (4) The cell wall-breaking liquid is subjected to low-temperature centrifugation to separate the supernatant and obtain crude enzyme solution; (5) The crude enzyme solution is subjected to two stages of ammonium sulfate precipitation and dialysis desalting to obtain SOD active components; in the two stages of ammonium sulfate precipitation, the first stage is 35% to 40% saturation and the second stage is 70% to 80% saturation. (6) Add glycine, trehalose and mannitol to the SOD active component for stabilization treatment, adjust the pH to 7.0 to 7.5, and let it stand at 4°C for 2 to 12 hours to obtain the SOD product; the amount of glycine added is 0.3% to 0.6%, the amount of trehalose added is 2.0% to 4.0%, and the amount of mannitol added is 1.0% to 2.0%.

2. The method for preparing superoxide dismutase from *Saccharomyces cerevisiae* according to claim 1, characterized in that, The freeze-thaw treatment in step (3) is performed 3 times, the glass beads have a particle size of 0.5 mm, the mass-to-volume ratio of glass beads to bacterial suspension is 0.5:1, and the shaking time is 20 min.

3. The method for preparing superoxide dismutase from *Saccharomyces cerevisiae* according to claim 1, characterized in that, The glass bead oscillation and cell wall breaking process in step (3) is carried out in an ice bath or at 4°C.

4. The method for preparing superoxide dismutase from *Saccharomyces cerevisiae* according to claim 1, characterized in that, The low-temperature centrifugation separation conditions in step (4) are: centrifugation temperature 2-8℃, centrifugation speed 8000-12000r / min, centrifugation time 10-25min; after centrifugation, further filtration and clarification are carried out using a 0.45 μm microporous membrane.

5. The method for preparing superoxide dismutase from *Saccharomyces cerevisiae* according to claim 1, characterized in that, The salt-precipitate obtained in step (5) is reconstituted with Tris-HCl buffer or phosphate buffer, wherein the pH of the reconstitution buffer is 7.0 to 8.0; the dialysis desalination in step (5) is performed using a dialysis bag with a molecular weight cutoff of 8kDa to 14kDa, dialyzed at 4°C for 8 to 24 hours, and the solution is changed 2 to 4 times during the dialysis process.

6. The method for preparing superoxide dismutase from *Saccharomyces cerevisiae* according to claim 1, characterized in that, In step (6), the amount of glycine added is 0.5%, the amount of trehalose added is 3.0%, and the amount of mannitol added is 1.5%. After adjusting the pH to 7.2, the mixture is allowed to stand at 4°C for 4 hours to reach equilibrium.

7. The method for preparing superoxide dismutase from *Saccharomyces cerevisiae* according to claim 1, characterized in that, In step (6), the amount of glycine added is 0.3%, the amount of trehalose added is 4.0%, and the amount of mannitol added is 2.0%. After adjusting the pH to 7.2, the mixture is allowed to stand at 4°C for 4 hours to reach equilibrium.

8. The method for preparing superoxide dismutase from *Saccharomyces cerevisiae* according to claim 1, characterized in that, The SOD product is a liquid or solid preparation; when it is a liquid preparation, it should be stored at 2–8°C away from light.

9. The SOD product prepared by the method according to any one of claims 1 to 8, characterized in that, The SOD product, after being diluted or reconstituted according to the testing requirements specified in GB / T 41906-2022, had an SOD enzyme activity of 900–1100 U / mL.

10. The SOD product according to claim 9, characterized in that, The enzyme activity retention rate of the SOD product was 84%–91% after being stored at 4°C for 6 months.

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