A method for extracting polysaccharides from *Micrococcus pluvialis* and its applications

Abstract

This invention discloses a method for extracting polysaccharides from *Microcystis aeruginosa* and its applications. The method includes fat removal, alcohol-soluble molecule removal, ultrasonic water extraction, flavonoid removal, protein removal, pigment removal, ethanol precipitation, small molecule substance removal, and freeze-drying. This invention combines directional stepwise impurity removal with ultrasonic water extraction to achieve efficient separation of polysaccharides from impurities. The optimal ultrasonic extraction process was determined using response surface methodology: a fixed ultrasonic extraction power of 100W, an ultrasonic extraction temperature of 49℃, an extraction time of 61 min, a solid-liquid ratio of 1:20.5 g / mL, and three extractions. Under these conditions, the polysaccharide yield reached 1.45%. The obtained *Microcystis aeruginosa* polysaccharides exhibit significant antioxidant activity, showing good scavenging effects against DPPH free radicals, hydroxyl free radicals, and ABTS free radicals, with the total reducing power increasing in a dose-dependent manner. This invention provides a simple and controllable extraction process, yielding polysaccharides with high purity and good activity, which can be used as natural antioxidants in functional foods, health products, and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of natural active ingredient extraction and biomedical technology, specifically to a method for extracting and applying polysaccharides from *Micrococcus pluvialis*. Background Technology

[0002] Polysaccharides, also known as polyunsaccharides, are high-molecular-weight polymers formed by the linear or branched linkage of aldoses or ketoses through glycosidic bonds. They are widely distributed in animal, plant, and microbial systems. As one of the most abundant biopolymers in nature, polysaccharides are also one of the four basic substances constituting life activities, and their unique structural characteristics endow them with diverse biological functions. Studies have confirmed that polysaccharides possess various biological activities such as immunomodulation, antitumor, antioxidant, and hypoglycemic effects. Moreover, as non-cytotoxic substances, they show great application potential in the research and development of functional foods and the biopharmaceutical field.

[0003] *Nannochloropsis gaditana*, belonging to the genus *Nannochloropsis* in the family Monocysticaceae, is a tiny, mostly green or yellowish-green marine food algae. In 2021, the National Health Commission of China officially approved *Nannochloropsis gaditana* as a new food ingredient (recommended daily intake ≤ 2 grams, based on dry weight), thus officially recognizing its application prospects in the food industry. This algae is rich in nutrients such as protein, eicosapentaenoic acid (EPA), and polysaccharides, possessing both good food safety and development value. However, current research on *Nannochloropsis gaditana* resources mainly focuses on lipid accumulation for the production of EPA or biofuels, while research on its polysaccharide extraction processes and activity is relatively lagging. Furthermore, the polysaccharides within *Chlorella pseudochlorella* cells are tightly bound to impurities such as fats, flavonoids, pigments, and proteins. Existing extraction and purification processes lack targeted design and generally suffer from low extraction efficiency, incomplete impurity removal, and easily compromised polysaccharide activity. No dedicated process exists that can simultaneously achieve high yield, high purity, and high bioactivity, severely hindering the in-depth development of *Chlorella pseudochlorella* polysaccharides in the pharmaceutical, health product, and food industries. Therefore, developing targeted and efficient extraction and purification methods for *Chlorella pseudochlorella* polysaccharides is of significant practical importance for fully exploring the industrial application potential of this new food ingredient. Summary of the Invention

[0004] This invention aims to provide a method for extracting polysaccharides from *Microcera pseudochlorella* and its applications, addressing the problems of low extraction efficiency, incomplete impurity removal, and easily compromised polysaccharide activity in existing extraction and purification processes. Through targeted and efficient impurity removal and extraction steps, the method achieves highly efficient separation of polysaccharides from impurities such as fats, flavonoids, pigments, and proteins. This improves yield and purity while maximizing the preservation of its natural biological activity, providing technical support for the industrial development of *Microcera pseudochlorella* as a novel food ingredient in functional foods, health products, and biomedicine.

[0005] To achieve the above objectives, the present invention is implemented through the following solution: A method for extracting polysaccharides from *Micrococcus pluvialis* includes the following steps: S1. Fat removal; S2, removal of alcohol-soluble molecules; S3, ultrasonic water extraction; S4, Flavonoid removal; S5, protein removal; S6. Pigment removal; S7, Ethanol precipitation; S8. Removal of small molecules; S9, freeze-drying.

[0006] Preferably, in step S1, the freeze-dried powder of *Micrococcus pseudocarpa* is weighed, and petroleum ether is added at a material-to-liquid ratio of 1:5-10 g / mL. The mixture is heated under reflux in a water bath at 50-60°C for 2-3 hours. This operation is repeated 3-5 times until the fat is completely removed. The defatted algae powder is then placed in an oven and dried at 50-60°C for later use.

[0007] Preferably, in step S2, the defatted *Micrococcus pseudocarpa* powder obtained in step S1 is taken and 95% ethanol is added at a material-to-liquid ratio of 1:10-15 g / mL. The mixture is then refluxed at 80°C for 2-3 hours and the extraction is repeated 2-3 times to initially remove pigments and other alcohol-soluble impurities. After the extraction system has cooled naturally, it is centrifuged at 3000 r / min for 15 minutes. The precipitate of *Micrococcus pseudocarpa* powder after centrifugation is collected and placed in an oven to dry at 50-60°C for later use.

[0008] Preferably, in step S3, the dried algae powder obtained in step S2 is added to a solvent and subjected to ultrasonic water extraction; the ultrasonic extraction power is fixed at 100W, the ultrasonic extraction temperature is controlled at 40-80℃, the extraction time is 20-100min, the material-to-liquid ratio is 1:10-30g / mL, and the number of extractions is 1-5 times; the solvent is deionized water.

[0009] Preferably, in step S4, the ultrasonic extracts obtained in step S3 are combined and concentrated to 1 / 3-1 / 5 of the original volume by rotary evaporation to obtain a concentrated solution; the concentrated solution is added to D101 macroporous adsorption resin at a material-to-liquid ratio of 1:10-15 g / mL and placed in a shaker for 8-12 hours to remove flavonoids from the extract.

[0010] Preferably, in step S5, the extract after flavonoid removal in step S4 is centrifuged and filtered to remove impurities and precipitates; then, an equal amount of Sevage reagent is added to the clarified filtrate, and after thorough mixing, the mixture is allowed to stand and separate into layers to remove the upper protein impurities. This process is repeated 5-10 times to complete the protein removal.

[0011] Preferably, in step S6, the clarified filtrate after protein removal in step S5 is taken and AB-8 or ADS-7 macroporous adsorption resin is added at a material-to-liquid ratio of 1:10-20 g / mL. The mixture is then placed in a shaker and shaken for 8-12 hours to further remove the remaining pigment impurities in the extract. After adsorption is complete, the filtrate is collected, which is the crude polysaccharide extract after pigment removal.

[0012] Preferably, in step S7, the depigmented crude polysaccharide extract obtained in step S6 is placed in a low-temperature environment and cooled to 4-8°C. 95% ethanol, pre-cooled to below 4°C, is slowly added while stirring until the volume fraction of ethanol in the system reaches 70%-90%. After standing and precipitating for 12-24 hours, the precipitate is centrifuged at 4000 r / min for 20 minutes and collected to obtain the crude polysaccharide product.

[0013] Preferably, in step S8, the crude polysaccharide obtained in step S7 is reconstituted with deionized water to obtain a polysaccharide reconstituted solution; the polysaccharide reconstituted solution is placed in a dialysis bag with a molecular weight cutoff of 3500-5000 Da, and dialyzed in deionized water. The dialysis temperature is controlled at 4-10℃, and the deionized water is replaced every 4-6 hours. Dialysis is performed for 48-72 hours until the dialysate shows no positive reaction when tested by the phenol-sulfuric acid method, thus completing the removal of small molecules; the polysaccharide solution in the dialysis bag is collected.

[0014] Preferably, in step S9, the polysaccharide solution obtained in step S8 after the removal of small molecules is placed in a vacuum freeze dryer for vacuum freeze drying; the freeze drying conditions are: pre-freezing temperature -40 to -80℃, pre-freezing time 2-3h, vacuum degree 0.01-0.03MPa, sublimation drying temperature -20 to -10℃, desorption drying temperature 20-30℃, and total drying time 24-48h; after drying, the high-quality polysaccharide of *Micrococcus pseudocarpa* is obtained, which is then pulverized and sealed for storage.

[0015] This invention also provides the application of the *Micrococcus pseudocarpa* polysaccharide extracted by the above method in the preparation of antioxidant products.

[0016] Furthermore, the polysaccharide from *Micrococcus pluvialis* has the ability to scavenge DPPH free radicals, hydroxyl free radicals, and ABTS free radicals, and has a strong total reducing power. It can be added as an active ingredient to antioxidant foods, health products, or cosmetics.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention designs a targeted stepwise impurity removal and extraction process based on the characteristics of the components of *Chlorella pseudomicrophylla*, which removes fat, alcohol-soluble impurities, flavonoids, proteins, pigments and small molecules in sequence. The impurities are removed more thoroughly, effectively improving the purity of *Chlorella pseudomicrophylla* polysaccharides and solving the problem of excessive impurity residue in existing processes.

[0018] (2) The present invention uses ultrasonic water extraction and optimizes extraction parameters to improve polysaccharide dissolution efficiency and increase yield, while controlling mild extraction and purification conditions throughout the process to maximize the preservation of the natural biological activity of polysaccharides and ensure their antioxidant and other functional properties.

[0019] (3) The extraction method of the present invention has clear process steps, well-defined parameters and strong controllability. The operation of each step is highly adaptable and can stably prepare high-quality polysaccharides of *Micrococcus pseudocarpa* with excellent antioxidant properties. These polysaccharides can effectively scavenge DPPH free radicals, hydroxyl free radicals and ABTS free radicals and have strong total reducing power. They can be directly used as active ingredients in the research and development and production of antioxidant functional foods, health products and cosmetics. This provides reliable technical support for the industrialization of *Micrococcus pseudocarpa* as a new food raw material in the field of antioxidants and has significant practical application value. Attached Figure Description

[0020] To make the technical solutions in the embodiments of the present invention or the prior art clearer, a brief description will be given below in conjunction with the accompanying drawings required in the embodiments. Obviously, the following drawings are only some embodiments of the present invention, and those skilled in the art can obtain other related drawings based on these drawings without creative effort.

[0021] Figure 1 This is a process flow diagram for extracting polysaccharides from *Micrococcus pseudocarpa* according to the present invention.

[0022] Figure 2 This is a standard curve for glucose.

[0023] Figure 3 The figure shows the results of single-factor experiments on the extraction of polysaccharides from *Microcera pseudocaryophylla* in this invention; where A represents the effect of ultrasonic extraction temperature on the yield of *Microcera pseudocaryophylla* polysaccharides; B represents the effect of extraction time on the yield of *Microcera pseudocaryophylla* polysaccharides; C represents the effect of the material-to-liquid ratio on the yield of *Microcera pseudocaryophylla* polysaccharides; and D represents the effect of the number of extractions on the yield of *Microcera pseudocaryophylla* polysaccharides.

[0024] Figure 4These are 2D contour plots and 3D response surface plots showing the results of extracting polysaccharides from *Microcystis aeruginosa* according to this invention. Specifically, A is a 2D contour plot of ultrasonic extraction temperature versus extraction time; B is a 3D response surface plot of ultrasonic extraction temperature versus extraction time; C is a 2D contour plot of ultrasonic extraction temperature versus material-to-liquid ratio; D is a 3D response surface plot of ultrasonic extraction temperature versus material-to-liquid ratio; E is a 2D contour plot of extraction time versus material-to-liquid ratio; and F is a 3D contour plot of extraction time versus material-to-liquid ratio.

[0025] Figure 5 The graph shows the evaluation results of the antioxidant capacity of the polysaccharide extracted from *Micrococcus pseudocarpa* obtained in this invention; where A is the DPPH free radical scavenging rate, B is the hydroxyl free radical scavenging rate, C is the ABTS free radical scavenging rate, and D is the total reducing capacity. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are only for explaining the present invention and are not intended to limit its scope of protection. Experimental methods not specified with specific conditions in the embodiments generally use conventional conditions in the art; materials or reagents used, unless otherwise stated, are all commercially available conventional products. It should be noted that the embodiments described herein are only some embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] Example 1: A method for extracting polysaccharides from *Microsporum simulans* S1. Fat removal; S2, removal of alcohol-soluble molecules; S3, ultrasonic water extraction; S4, Flavonoid removal; S5, protein removal; S6. Pigment removal; S7, Ethanol precipitation; S8. Removal of small molecules; S9, freeze-drying.

[0028] The following details each step. The ultrasonic water extraction step uses single-factor experiments combined with response surface methodology to optimize and determine the optimal process parameters. All other steps are implemented using fixed process parameters.

[0029] 1. Fat removal Weigh out the freeze-dried powder of Micrococcus pseudochlorella, add petroleum ether at a ratio of 1:8 g / mL, heat and reflux in a 55℃ water bath for 2.5 h, repeat the operation 4 times until the fat is completely removed; dry the defatted algae powder in a 55℃ oven, cool and seal for later use.

[0030] 2. Removal of alcohol-soluble molecules Take the defatted *Micrococcus pseudoglobulus* powder obtained in step 1, add 95% ethanol at a material-to-liquid ratio of 1:12 g / mL, reflux extract at 80℃ for 2.5 h, and repeat the extraction twice; after the extraction system has cooled naturally, centrifuge at 3000 r / min for 15 min, collect the algal powder precipitate, and dry it in an oven at 55℃ for later use.

[0031] 3. Ultrasonic water extraction Using deionized water as the extraction solvent, the dried algae powder obtained in step 2 was subjected to ultrasonic water extraction with a fixed ultrasonic power of 100W. First, single-factor experiments were conducted to investigate the effects of ultrasonic extraction temperature, extraction time, material-to-liquid ratio, and number of extractions on the polysaccharide yield. Then, based on the results of the single-factor experiments, response surface methodology was used to optimize the key process parameters and determine the optimal ultrasonic water extraction conditions.

[0032] 3.1 Polysaccharide yield determination The polysaccharide yield was determined using the following method: Anhydrous glucose solution was used as a standard control, and the polysaccharide yield was determined using the phenol-sulfuric acid method. Anhydrous glucose was weighed and a 100 μg / mL glucose solution was prepared. This solution was then diluted to concentrations of 12.5, 25, 37.5, 50, 62.5, 75, 87.5, and 100 μg / mL. 400 μL of each glucose solution was added to a reaction tube, followed by 200 μL of 6% phenol and 1 mL of concentrated sulfuric acid. The mixture was thoroughly mixed and reacted at 90 °C for 10 min. After cooling to room temperature, the absorbance of each well was measured at a wavelength of 490 nm. A standard curve was plotted with the glucose solution concentration (μg / mL) on the x-axis and the absorbance on the y-axis. Weigh an appropriate amount of *Micrococcus pseudochlorella* polysaccharide and prepare a 0.1 mg / mL solution. Other procedures are the same as for preparing the standard curve. Measure the absorbance of the sample and substitute it into the standard curve equation to obtain the polysaccharide concentration. The polysaccharide yield is calculated using equation (1).

[0033] Polysaccharide yield = (ρ×N×V) / m×100% (1) In the formula: ρ is the mass concentration of polysaccharide in the sample solution, mg / mL; N is the dilution factor; V is the volume of polysaccharide solution, mL; and m is the mass of lyophilized Micrococcus pseudocarpa powder, g.

[0034] 3.2 Single-factor experimental design Using polysaccharide yield as an indicator, the following method was used: 2g of *Micrococcus pseudocarpa* powder from step S2 was accurately weighed. Extraction was performed at different ultrasonic extraction temperatures (40, 50, 60, 70, 80℃), different ultrasonic extraction times (20, 40, 60, 80, 100 min), different material-to-liquid ratios (1:10, 1:15, 1:20, 1:25, 1:30 g / mL), and different extraction times (1, 2, 3, 4, 5 times) according to the set power. Specific operation and condition settings are as follows: (1) Accurately weigh 2g of *Micrococcus pseudocarpa* powder from step S2, set the ultrasonic extraction power to 100W, set the material-liquid ratio to 1:20g / mL, the extraction time to 60min, the number of extractions to 3, and the temperature gradient to 40℃, 50℃, 60℃, 70℃, and 80℃.

[0035] (2) Accurately weigh 2g of *Micrococcus pluvialis* powder from step S2, set the ultrasonic extraction power to 100W, set the extraction temperature to 50℃ (select the extraction temperature with the highest yield based on the experimental results of the extraction temperature), the material-liquid ratio to 1:20g / mL, extract 3 times, and the time gradient to 20min, 40min, 60min, 80min, and 100min. (3) Accurately weigh 2g of Micrococcus pseudocarpa powder in step S2, set the ultrasonic extraction power to 100W, set the extraction temperature to 50℃ (select the extraction temperature with the highest yield according to the extraction temperature experiment results), the extraction time to 60min, the number of extractions to 3, and the material-liquid ratio gradient to be 1:10, 1:15, 1:20, 1:25, 1:30g / mL. (4) Accurately weigh 2g of *Micrococcus pseudocarpa* powder from step S2, set the ultrasonic extraction power to 100W, set the extraction temperature to 50℃ (select the extraction temperature with the highest yield based on the experimental results of the extraction temperature), the extraction time to 60min, the material-liquid ratio to 1:20g / mL, and the extraction times to 1, 2, 3, 4, and 5 times.

[0036] 3.3 Response Surface Design Based on the results of the single-factor experiments, ultrasonic extraction temperature, extraction time, and solid-liquid ratio, which significantly affected polysaccharide yield, were selected as independent variables in the response surface methodology. Polysaccharide yield was used as the response value. A Box-Behnken experimental design was employed for a three-factor, three-level experiment. Regression equations were fitted, the interactions between factors were analyzed, and the optimal process parameters for ultrasonic water extraction were determined. The number of ultrasonic extractions was determined to be three based on the single-factor experimental results, and the ultrasonic power was fixed at 100W throughout the process. The response surface methodology factors and levels are shown in Table 1.

[0037] Table 1. Factors and levels in response surface methodology experiment 4. Flavonoid removal The combined ultrasonic extract obtained in step 3 was concentrated to 1 / 4 of its original volume by rotary evaporation to obtain a polysaccharide concentrate. D101 macroporous adsorption resin was added to the concentrate at a material-to-liquid ratio of 1:13 g / mL. The concentrate was placed in a shaker and shaken for 10 hours at a shaking speed of 135 r / min and a temperature of 27℃ to remove flavonoids. The adsorbed extract was collected for later use.

[0038] 5. Protein removal Centrifuge the extract after flavonoid removal in step 4 (3500 r / min, 15 min), filter to remove impurities and precipitates; add an equal amount of Sevage reagent (chloroform and n-butanol mixed at a volume ratio of 4:1) to the clear filtrate, mix thoroughly, allow to stand and separate into layers, remove the lower layer of protein impurities, repeat this operation 5-10 times until the protein is completely removed, and collect the upper aqueous phase filtrate for later use.

[0039] 6. Pigment removal Take the clear filtrate after protein removal in step 5, add AB-8 macroporous adsorption resin at a material-to-liquid ratio of 1:15 g / mL, place it in a shaker, and shake and adsorb for 10 hours at a shaking speed of 135 r / min and a temperature of 27℃ to further remove the remaining pigment impurities in the filtrate; after adsorption is complete, filter and collect the filtrate, which is the crude polysaccharide extract after pigment removal.

[0040] 7. Ethanol precipitation The crude polysaccharide extract obtained in step 6 was placed in a low-temperature environment and cooled to 4°C. 95% ethanol, which had been pre-cooled to below 4°C, was slowly added to it while stirring at a constant speed until the volume fraction of ethanol in the system reached 80%. After standing at room temperature for 12-24 hours to precipitate, the precipitate was centrifuged at 4000 r / min for 20 min and collected. This precipitate is the crude polysaccharide product of *Micrococcus pseudocarpa*.

[0041] 8. Removal of small molecules The crude polysaccharide obtained in step S7 was fully reconstituted with deionized water to obtain a polysaccharide reconstituted solution. The polysaccharide reconstituted solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, sealed, and placed in deionized water for dialysis. The dialysis temperature was controlled at 4-10℃, and the deionized water was changed every 4-6 hours. Dialysis was continued for 48-72 hours until the dialysate showed no positive reaction when tested by the phenol-sulfuric acid method, indicating that the small molecule substances were completely removed. The polysaccharide solution in the dialysis bag was collected for later use.

[0042] 9. Freeze-drying The polysaccharide solution obtained in step 8 after the removal of small molecules was placed in a vacuum freeze dryer and subjected to vacuum freeze drying under the following conditions: pre-freezing temperature -40 to -80℃, pre-freezing time 2-3h, vacuum degree 0.01-0.03MPa, sublimation drying temperature -20 to -10℃, desorption drying temperature 20-30℃, and total drying time 24-48h. After drying, the high-quality polysaccharide of *Micrococcus pseudocarpa* was obtained, which was then pulverized and sealed for storage.

[0043] 10. Experimental Results 10.1 Overall Process Flow The polysaccharide extracted by this invention and the overall process flow are as follows: Figure 1 As shown, the extracted *Micrococcus pseudochlorella* polysaccharide is a yellowish-white powder that is easily soluble in water but insoluble in organic solvents such as ethanol.

[0044] 10.2 Glucose Standard Curve Depend on Figure 2 It can be seen that the linear regression equation between the absorbance value (y) and the mass concentration (x) of the glucose standard solution is y=0.0087x+0.1452, R²=0.9973, indicating that glucose has a good linear relationship within the concentration range set in the experiment, with a correlation coefficient close to 1 and a high degree of linear fit. This meets the experimental requirements for subsequent determination of the polysaccharide content and extraction rate of *Micrococcus pseudocarpa*, and can be used as a quantitative basis for data analysis in subsequent experiments.

[0045] 10.3 Results of Single-Factor Experiment 10.3 Analysis of Single-Factor Experiment Results 10.3.1 Effect of ultrasonic extraction temperature on the yield of *Micrococcus pseudocarpa* polysaccharides Depend on Figure 3 As shown in Figure A, the yield of *Micrococcus pseudochlorella* polysaccharides initially increased and then decreased with increasing ultrasonic extraction temperature, reaching a maximum of 1.40% at 50℃, significantly higher than other temperature groups (P<0.05). At 40℃, the yield was 1.29%. When the temperature increased to 50℃, the combined effect of ultrasound and heat promoted polysaccharide dissolution, significantly increasing the yield. However, when the temperature exceeded 50℃, the polysaccharide yield gradually decreased, dropping to 1.19% at 80℃. This may be due to the degradation or structural damage of polysaccharide molecules caused by high temperatures, which also promoted the dissolution of impurities, thus reducing the polysaccharide yield. Therefore, the ultrasonic extraction temperature (40–60℃) was selected for subsequent response surface methodology optimization experiments.

[0046] 10.3.2 Effect of extraction time on the yield of *Micrococcus pseudocarpa* polysaccharides like Figure 3As shown in Figure B, the polysaccharide yield first increased and then decreased with prolonged extraction time, reaching its highest level (1.40%) at 60 min, significantly higher than other time groups (P<0.05). At 20 min, the yield was only 1.14%. As extraction time increased to 60 min, intracellular polysaccharides were fully dissolved, and the yield gradually increased. After 60 min, the yield slowly decreased, reaching 1.27% at 100 min. This may be because prolonged ultrasonic treatment led to polysaccharide structural breakage and increased impurity dissolution, thus reducing the polysaccharide yield. Therefore, extraction time (40–80 min) was selected for subsequent response surface methodology optimization experiments.

[0047] 10.3.3 Effect of material-to-liquid ratio on the yield of *Micrococcus pseudocarpa* polysaccharides Depend on Figure 3 As shown in Figure C, the polysaccharide yield initially increased and then decreased with increasing solid-liquid ratio, reaching a peak of 1.45% at a solid-liquid ratio of 1:20 g / mL, significantly higher than other groups (P<0.05). At a solid-liquid ratio of 1:10 g / mL, the yield was only 1.15%, indicating insufficient solvent limiting polysaccharide dissolution. As the solid-liquid ratio increased to 1:20 g / mL, the increased solvent volume facilitated sufficient polysaccharide diffusion and dissolution, significantly improving the yield. Further increasing the solid-liquid ratio to 1:30 g / mL resulted in a slight decrease in yield to 1.35%, likely due to excessive dilution leading to a lower polysaccharide concentration and increased losses during subsequent concentration. Therefore, solid-liquid ratios of (1:15–1:25 g / mL) were selected for subsequent response surface methodology optimization experiments.

[0048] 10.3.4 Effect of extraction times on the yield of *Micrococcus pseudocarpa* polysaccharides like Figure 3 As shown in Figure D, the polysaccharide yield initially increased significantly with the number of extractions, reaching a maximum of 1.43% at 3 extractions, which was significantly higher than the 1- and 2-extraction groups (P<0.05). The yield was only 1.13% at 1 extraction. As the number of extractions increased to 3, residual polysaccharides within the cells were gradually and completely extracted, resulting in a significant increase in yield. After more than 3 extractions, the yield tended to stabilize, reaching 1.40% and 1.37% at 4 and 5 extractions, respectively, with no significant difference from the 3-extraction group (P>0.05). This indicates that 3 extractions were sufficient to achieve complete dissolution of the polysaccharides, and further increasing the number of extractions did not significantly contribute to improving the yield.

[0049] 10.4 Results of response surface methodology and analysis of variance 10.4.1 Response Surface Experiment Results As shown in the results in 10.3, the optimal number of extractions is 3. However, the optimal parameters for the other process variables have not yet been determined. Therefore, the Design-Expert 13.0 software was used to conduct response surface optimization experiments. The specific experimental results are shown in Table 2.

[0050] Table 2 Results of response surface methodology experiments serial number A (°C) B (min) C (g / mL) Yield of Micrococcus pseudochlorella polysaccharides (%) 1 50 60 20 1.42 2 60 80 20 1.27 3 40 60 25 1.19 4 60 60 25 1.10 5 40 80 20 1.13 6 50 60 20 1.43 7 50 40 15 1.04 8 40 60 15 1.11 9 50 60 20 1.41 10 60 60 15 1.03 11 40 40 20 1.24 12 50 80 25 1.14 13 50 80 15 1.16 14 50 40 25 1.17 15 50 60 20 1.44 16 50 60 20 1.39 17 60 40 20 1.07 10.4.2 Regression Equation and Analysis of Variance Results Based on software analysis, the quadratic regression model equation with the yield of *Micrococcus pseudochlorella* polysaccharide (Y) as the objective function is obtained as follows: Y = 1.418 - 0.0250A + 0.0225B + 0.0325C + 0.0775AB - 0.0025AC - 0.0375BC - 0.13025A 2 -0.11025B 2 -0.18025C 2 .

[0051] ANOVA was performed on the quadratic response surface methodology (ANOVA) model established for the polysaccharide yield of *Microcystis aeruginosa*. The results are shown in Table 3. The model's F-value was 63.14, P < 0.0001, reaching a highly significant level, indicating that the model can effectively reflect the relationship between various experimental factors and polysaccharide yield, and that only 0.01% of the error is due to random error. The lack-of-fit term had a P > 0.05, which was not significant, indicating that the model fits the experimental data well and there is no obvious systematic bias. It can be used for prediction and optimization of subsequent extraction processes.

[0052] The model's coefficient of determination R² = 0.9878, and the adjusted coefficient of determination R... adj R² = 0.9722 and the predictive coefficient of determination. pred The value of 2² = 0.8683 is quite close (difference less than 0.2), indicating that the model not only has a high fit but also good predictive ability. The signal-to-noise ratio (AdeqPrecision) is 20.559, much greater than the critical value of 4, further confirming that the model has sufficient resolution and reliability. In addition, the low coefficient of variation (CV% = 1.99%) indicates high experimental accuracy and good data repeatability. Therefore, this model can be used for the optimization analysis of the extraction process of polysaccharides from *Chlorella vulgaris*.

[0053] In the first-order terms, the material-to-liquid ratio (C), ultrasonic extraction temperature (A), and extraction time (B) all significantly affected the polysaccharide yield of *Chlorella vulgaris* (P<0.05). Based on the F-values, the degree of influence of each factor was in the order of material-to-liquid ratio > ultrasonic extraction temperature > extraction time. In the interaction terms, the interactions between ultrasonic extraction temperature and extraction time (AB, P<0.01) and extraction time and material-to-liquid ratio (BC, P<0.05) were both significant, while the interaction between ultrasonic extraction temperature and material-to-liquid ratio (AC, P>0.05) was not significant. This indicates that the synergistic effect of temperature and time, and time and material-to-liquid ratio, significantly affects the polysaccharide yield. In the second-order terms, A², B², and C² were all highly significant (P<0.01), indicating that the relationship between each factor and the polysaccharide yield is not a simple linear one, but rather exhibits a clear quadratic effect, meaning that the yield shows a parabolic trend of first increasing and then decreasing with the change in the levels of each factor.

[0054] Table 3. Analysis of Variance in Response Surface Experiments Note: "**" indicates extremely significant difference (P<0.01), "*" indicates significant difference (P<0.05), and "ns" indicates no significant difference (P>0.05).

[0055] 10.4.3 Response Surface Interaction Analysis 2D contour plots can visually reflect the strength of the interaction between two factors, while the steepness of the 3D response surface can characterize the degree of influence of each factor on the yield of *Micrococcus pseudocarpa* polysaccharides. Figure 4 It is evident that all response surface features a downward-opening convex shape, indicating a maximum point in the polysaccharide yield. Among these, the ultrasonic extraction temperature and extraction time... Figure 4 A, B), Extraction time and solid-liquid ratio ( Figure 4 The interaction between E and F is significant, with their 2D contour lines exhibiting a compact elliptical distribution and the 3D response surface displaying a distinct "first rise, then fall" steep curve, indicating a strong synergistic effect between the two factors; while the ultrasonic extraction temperature and the material-liquid ratio ( Figure 4 The contour lines of C and D are relatively flat and nearly circular, and the 3D surface undulations are small, indicating that the corresponding interaction is not significant. This result is consistent with the conclusion of the analysis of variance.

[0056] The optimal extraction process for *Chlorella vulgaris* polysaccharides was determined through response surface methodology (RSM) optimization experiments. The optimal RSM was found to be an ultrasonic extraction temperature of 49.204℃, an extraction time of 61.196 min, and a solid-liquid ratio of 1:20.422 g / mL, resulting in an extraction rate of 1.42%. Considering the feasibility of each factor, the above conditions were further optimized to an ultrasonic extraction temperature of 49℃, an extraction time of 61 min, and a solid-liquid ratio of 1:20.5. Three repeated validation experiments were conducted under these conditions, yielding an average polysaccharide yield of 1.45%, which is close to the predicted value. This indicates that the model is reasonable and feasible and can be used for optimizing and predicting the extraction process of *Chlorella vulgaris* polysaccharides.

[0057] Example 2: Antioxidant activity analysis of the *Micrococcus pseudocarpa* polysaccharide extracted in this invention. 1. Assay of DPPH free radical scavenging activity Prepare sample solutions of *Micrococcus pseudochlorella* polysaccharide at concentration gradients of 0.125, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, and 4.00 mg / mL. Take 1.0 mL of the sample solution and mix thoroughly with 1.0 mL of 0.15 mM DPPH (prepared with 50% ethanol solution). React at room temperature in the dark for 30 min, and measure the absorbance of the system at 517 nm, denoted as A1. Replace the DPPH-ethanol solution with 50% ethanol and measure the absorbance, denoted as A2. Replace the sample solution with 50% ethanol and measure the absorbance, denoted as A3. Use ascorbic acid (Vc) as a positive control, and calculate the DPPH free radical scavenging rate according to Formula 1.

[0058] DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A3] × 100% (1) 2. Hydroxyl radical scavenging activity assay Prepare sample solutions of *Micrococcus pseudochlorella* polysaccharide at concentrations of 0.125, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, and 4.00 mg / mL. Take 1.0 mL of the sample solution and add 1.0 mL of 1.5 mM FeSO4 solution, 0.7 mL of 6.0 mM H2O2 solution, and 0.3 mL of 20 mM salicylic acid solution sequentially. After shaking and mixing, incubate at 37℃ for 1 h. Measure the absorbance of the system at 562 nm and record it as A1. Replace the salicylic acid solution with ultrapure water and measure the absorbance, recording it as A2. Use ultrapure water instead of the polysaccharide solution as a blank control and measure the absorbance, recording it as A3. Use ascorbic acid (Vc) as a positive control and calculate the hydroxyl radical scavenging rate according to Formula 2.

[0059] Hydroxyl radical scavenging rate (%) = [1 - (A1 - A2) / A3] × 100% (2) 3. Assay of ABTS free radical scavenging activity First, prepare the ABTS radical working solution: Mix 7.0 mM ABTS solution with 2.45 mM potassium persulfate solution and react at room temperature in the dark for 16 h. Dilute with 0.2 M, pH 7.4 PBS solution to obtain an absorbance of 0.70 ± 0.02 at 734 nm. Take 20 μL of *Microcystis aeruginosa* polysaccharide sample solutions with concentrations of 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, and 4.00 mg / mL, add them to 200 μL of the above ABTS radical working solution in a 96-well plate, mix thoroughly, and react at room temperature for 6 min. Measure the absorbance at 734 nm and record it as A1. Replace the ABTS working solution with 200 μL of 0.2 M, pH 7.4 PBS solution and measure the absorbance as A2. Replace the sample solution with 20 μL of ultrapure water and measure the absorbance as A3. Using ascorbic acid (Vc) as a positive control, the ABTS free radical scavenging rate was calculated according to Formula 3.

[0060] ABTS free radical scavenging rate (%) = [1 - (A1 - A2) / A3] × 100% (3) 4. Total reducing power determination Prepare sample solutions of *Micrococcus pseudochlorella* polysaccharides at concentrations of 0.125, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, and 4.00 mg / mL. Take 1 mL of the sample solution, add 1 mL of 0.2 M PBS solution (pH 6.6) and 1 mL of 1% K3[Fe(CN)6] solution, mix thoroughly, and heat in a 50℃ water bath for 20 min. Then add 1 mL of 10% trichloroacetic acid solution, centrifuge at 4000 r / min for 10 min, take 0.5 mL of the supernatant, add 0.1 mL of 0.1% FeCl3 solution and 0.5 mL of ultrapure water, mix well, let stand for 10 min, and measure the absorbance at 700 nm, recording it as A1. Replace the FeCl3 solution with ultrapure water and measure the absorbance, recording it as A2. Use ascorbic acid (Vc) as a positive control, and calculate the total reducing power of the sample according to Formula 4.

[0061] Total reducing force = A1 - A2 (4) 5. Experimental Results 5.1 DPPH free radical scavenging ability like Figure 5 As shown in Figure A, the scavenging ability of *Microcystis aeruginosa* polysaccharide against DPPH free radicals exhibits a significant concentration-dependent effect. Within the concentration range of 0.125–4.0 mg / mL, the scavenging rate continuously increases with increasing sample concentration: 15.91% at 0.125 mg / mL, 52.02% at 2.0 mg / mL, and 98.41% at 4.0 mg / mL, approaching the maximum scavenging level. The half-maximal inhibitory concentration (IC50) of *Microcystis aeruginosa* polysaccharide against DPPH free radicals was calculated.50 The concentration of the active ingredient was 1.85 mg / mL. The positive control, vitamin C, maintained high scavenging activity throughout the same concentration range, achieving a scavenging rate of 96.46% at 0.125 mg / mL, and remaining stable between 94% and 99% across the entire test concentration range. At low concentrations (≤1.5 mg / mL), the scavenging ability of *Micrococcus pseudochlorella* polysaccharide was significantly lower than that of vitamin C; however, as the concentration increased, the difference gradually narrowed, and at 4.0 mg / mL, its scavenging rate (98.41%) was essentially equal to that of vitamin C (97.17%).

[0062] 5.2 Hydroxyl radical scavenging ability The scavenging effect of *Micrococcus pseudocarpa* polysaccharides on hydroxyl radicals is as follows: Figure 5 As shown in Figure B, the scavenging rate gradually increased with increasing sample concentration, exhibiting a clear dose-response relationship. When the concentration increased from 0.125 mg / mL to 2.0 mg / mL, the scavenging rate increased from 35.74% to 96.71%; thereafter, with further increases in concentration, the scavenging rate tended to plateau, reaching 99.08% at 4.0 mg / mL, approaching complete scavenging. The half-maximal inhibitory concentration (IC50) of *Micrococcus pseudochlorella* polysaccharide against hydroxyl radicals was calculated. 50 The concentration of the active ingredient was 0.31 mg / mL. The positive control, vitamin C, showed a more significant hydroxyl radical scavenging ability, achieving a scavenging rate of 85.11% at 0.5 mg / mL and 99.53% at 1.0 mg / mL, after which it tended to stabilize. These results indicate that *Micrococcus pseudocarpa* polysaccharide possesses a strong hydroxyl radical scavenging ability, although slightly lower than that of vitamin C, it still exhibits good antioxidant potential.

[0063] 5.3 ABTS free radical scavenging ability The scavenging effect of *Micrococcus pseudocarpa* polysaccharide on ABTS free radicals is as follows: Figure 5 As shown in Figure C, the scavenging rate gradually increased with increasing sample concentration, exhibiting a clear concentration-dependent effect. In the lower concentration range (0.5-2.0 mg / mL), the scavenging rate increased relatively gradually, from 2.17% to 60.81%; however, when the concentration exceeded 2.0 mg / mL, the scavenging rate increased rapidly, reaching 83.56% at 3.0 mg / mL and 91.96% at 4.0 mg / mL. The half-maximal inhibitory concentration (IC50) of *Microcystis aeruginosa* polysaccharide against ABTS free radicals was calculated. 50 The concentration of the active ingredient was 1.53 mg / mL. The positive control, vitamin C, exhibited extremely strong ABTS radical scavenging ability within the same concentration range, with scavenging rates consistently above 98% at all tested concentrations (0.5-4.0 mg / mL), approaching complete removal. These results indicate that *Micrococcus pseudochlorella* polysaccharide possesses a certain ABTS radical scavenging ability in a concentration-dependent manner, but its scavenging effect is weaker than that of vitamin C.

[0064] 5.4 Overall Restoration Capacity The total reducing power of *Microcystis aeruginosa* polysaccharides was evaluated by measuring the difference in absorbance at different concentrations. The results are as follows: Figure 5 As shown in Figure D, the absorbance difference of *Chlorella vulgaris* polysaccharide gradually increased with increasing sample concentration, indicating that its reducing power was significantly concentration-dependent. Within the concentration range of 0.125–4.0 mg / mL, the absorbance difference of *Chlorella vulgaris* polysaccharide gradually increased from 0.46 to 1.19, showing a continuously enhancing reducing power. The absorbance value of the positive control, vitamin C, also increased with increasing concentration within the same concentration range, and remained consistently higher than that of *Chlorella vulgaris* polysaccharide, reaching 2.11 at 4.0 mg / mL. The results indicate that *Chlorella vulgaris* polysaccharide possesses a certain total reducing power, which is concentration-dependent, but its reducing power is weaker than that of vitamin C.

[0065] In summary, this invention establishes a targeted, stepwise impurity removal and ultrasonic water extraction process tailored to the compositional characteristics of *Micrococcus pseudochlorella*, a novel food ingredient. By sequentially removing fats, alcohol-soluble impurities, flavonoids, proteins, pigments, and small molecules, the technical challenge of tightly bound polysaccharides and impurities, making separation difficult, is effectively addressed. Based on this, response surface methodology was used to optimize the ultrasonic extraction temperature, extraction time, and solid-liquid ratio. The optimal process parameters were determined as follows: a fixed ultrasonic extraction power of 100W, an ultrasonic extraction temperature of 49℃, an extraction time of 61 min, a solid-liquid ratio of 1:20.5 g / mL, and three extractions. Under these conditions, the polysaccharide yield reached 1.45%, which highly matched the model prediction, verifying the stability and reliability of the process. In vitro antioxidant activity evaluation results showed that the *Microcystis aeruginosa* polysaccharides extracted in this invention exhibited significant scavenging effects against DPPH, hydroxyl, and ABTS free radicals, with half-maximal inhibitory concentrations (IC50) of 1.85 mg / mL, 0.31 mg / mL, and 1.53 mg / mL, respectively. They also demonstrated good total reducing power, with all exhibiting a clear dose-response relationship. Therefore, the *Microcystis aeruginosa* polysaccharides prepared in this invention have a simple and controllable extraction process, high purity, and significant antioxidant activity. They can serve as a high-quality source of natural antioxidants for functional foods, health products, and cosmetics, showing promising application development prospects.

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit its scope of protection. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for extracting polysaccharides from *Micrococcus pseudocarpa*, characterized in that: Includes the following steps: S1. Fat removal; S2, removal of alcohol-soluble molecules; S3, ultrasonic water extraction; S4, Flavonoid removal; S5, protein removal; S6. Pigment removal; S7, Ethanol precipitation; S8. Removal of small molecules; S9, freeze-drying.

2. The method for extracting polysaccharides from *Micrococcus pluvialis* as described in claim 1, characterized in that: In step S1, weigh the freeze-dried powder of *Micrococcus pseudocarpa*, add petroleum ether at a material-to-liquid ratio of 1:5-10 g / mL, and heat under reflux in a water bath at 50-60℃ for 2-3 hours. Repeat this operation 3-5 times until the fat is completely removed. Place the defatted algae powder in an oven and dry it at 50-60℃ for later use.

3. The method for extracting polysaccharides from *Micrococcus pluvialis* as described in claim 1, characterized in that: In step S2, the defatted Micrococcus pluvialis powder obtained in step S1 is taken and 95% ethanol is added at a material-to-liquid ratio of 1:10-15 g / mL. The mixture is then refluxed at 80°C for 2-3 hours and the extraction is repeated 2-3 times to initially remove pigments and other alcohol-soluble impurities. After the extraction system has cooled naturally, centrifuge at 3000 r / min for 15 min, collect the precipitate of *Micrococcus pseudocarpa* powder after centrifugation, place the powder in an oven and dry it at 50-60℃ for later use.

4. The method for extracting polysaccharides from *Micrococcus pluvialis* as described in claim 1, characterized in that: In step S3, the dried algae powder obtained in step S2 is added to a solvent and subjected to ultrasonic water extraction. The ultrasonic extraction power is fixed at 100W, the ultrasonic extraction temperature is controlled at 40-80℃, the extraction time is 20-100min, the material-to-liquid ratio is 1:10-30 g / mL, and the extraction is performed 1-5 times. The solvent is deionized water.

5. The method for extracting polysaccharides from *Micrococcus pluvialis* as described in claim 1, characterized in that: In step S4, the ultrasonic extracts obtained in step S3 are combined and concentrated to 1 / 3-1 / 5 of the original volume by rotary evaporation to obtain a concentrated solution. The concentrated solution is added to D101 macroporous adsorption resin at a material-to-liquid ratio of 1:10-15 g / mL and placed in a shaker for 8-12 hours to remove flavonoids from the extract.

6. The method for extracting polysaccharides from *Micrococcus pluvialis* as described in claim 1, characterized in that: In step S5, the extract after flavonoid removal in step S4 is centrifuged and filtered to remove impurities and precipitates. Then, an equal amount of Sevage reagent is added to the clarified filtrate, and after thorough mixing, the mixture is allowed to stand and separate into layers to remove the upper protein impurities. This process is repeated 5-10 times to complete the protein removal.

7. The method for extracting polysaccharides from *Micrococcus pseudocarpa* as described in claim 1, characterized in that: In step S6, the clarified filtrate after protein removal in step S5 is taken and AB-8 or ADS-7 macroporous adsorption resin is added at a material-to-liquid ratio of 1:10-20 g / mL. The mixture is then placed in a shaker and shaken for 8-12 hours to further remove the remaining pigment impurities in the extract. After adsorption is complete, the filtrate is collected, which is the crude polysaccharide extract after pigment removal.

8. The method for extracting polysaccharides from *Micrococcus pluvialis* as described in claim 1, characterized in that: In step S7, the depigmented crude polysaccharide extract obtained in step S6 is placed in a low-temperature environment and cooled to 4-8°C. 95% ethanol, pre-cooled to below 4°C, is slowly added while stirring until the volume fraction of ethanol in the system reaches 70%-90%. After standing and precipitating for 12-24 hours, the precipitate is centrifuged at 4000 r / min for 20 minutes and collected to obtain the crude polysaccharide product.

9. The method for extracting polysaccharides from *Micrococcus pluvialis* as described in claim 1, characterized in that: In step S8, the crude polysaccharide obtained in step S7 is reconstituted with deionized water to obtain a polysaccharide reconstituted solution. The polysaccharide reconstituted solution is placed in a dialysis bag with a molecular weight cutoff of 3500-5000 Da and dialyzed in deionized water. The dialysis temperature is controlled at 4-10℃, and the deionized water is replaced every 4-6 hours. Dialysis is performed for 48-72 hours until the dialysate shows no positive reaction when tested by the phenol-sulfuric acid method, thus completing the removal of small molecules. The polysaccharide solution in the dialysis bag is collected.

10. The method for extracting polysaccharides from *Micrococcus pluvialis* as described in claim 1, characterized in that: In step S9, the polysaccharide solution obtained in step S8 after the removal of small molecules is placed in a vacuum freeze dryer for vacuum freeze drying. The freeze drying conditions are: pre-freezing temperature -40 to -80℃, pre-freezing time 2-3h, vacuum degree 0.01-0.03MPa, sublimation drying temperature -20 to -10℃, desorption drying temperature 20-30℃, and total drying time 24-48h. After drying, the high-quality polysaccharide from *Micrococcus pluvialis* is obtained, which is then pulverized and sealed for storage.

11. The use of *Microcystis aeruginosa* polysaccharide extracted by the method according to any one of claims 1-10 in the preparation of products with antioxidant functions. The *Microcystis aeruginosa* polysaccharide has the ability to scavenge DPPH free radicals, hydroxyl free radicals, and ABTS free radicals, and has a strong total reducing power, and can be added as an active ingredient to antioxidant foods, health products, or cosmetics.

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