Method for extracting polysaccharide from selenium-rich rhodopseudomonas palustris fermentation liquor

Abstract

The invention discloses a method for extracting polysaccharide from selenium-rich rhodopseudomonas palustris fermentation broth, and belongs to the technical field of microbial fermentation engineering and biotechnology application. S2, decoloring: decoloring the primary concentrated solution by using a d-limonene and ethyl acetate mixed system under the action of aminated silicon dioxide nanoparticles to obtain a colorless and transparent polysaccharide solution; s3, precipitation and impurity removal: adding an organic acid salt composed of magnesium citrate and ferrous malate, stirring, adjusting the pH value to 4.5-5.5, reacting, and centrifuging to remove the precipitate; s4, desalting and purifying; and S5, drying. According to the method, the use of high-concentration ethanol is avoided in the whole process, the organic solvent residue in the product is extremely low, and multiple beneficial effects of zero alcohol precipitation, zero inorganic selenium residue, high polysaccharide yield and extremely high product purity are realized in the field of selenium-rich polysaccharide extraction for the first time; and reliable and irreplaceable technical support is provided for developing safe and efficient selenium-rich polysaccharide functional food.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation engineering and biotechnology application technology, and relates to a method for extracting polysaccharides from the fermentation broth of selenium-rich Rhodopseudomonas swampus. Background Technology

[0002] Selenium is one of the essential trace elements for the human body. The human body cannot synthesize selenium and must obtain it from food. Selenium in nature is mainly divided into two categories: inorganic and organic. Inorganic selenium is unsuitable as a source of selenium supplementation because of its high toxicity, poor absorption, and tendency to accumulate and be difficult to excrete. Organic selenium is the only biologically active form in the human body; therefore, selenium supplementation should be from organic sources. *Rhodopseudomonas palustris* (… Rhodopseudomonaspalustris ( ) is a photosynthetic bacterium that can convert inorganic selenium, such as sodium selenite, into active selenium through reductases, which is then integrated into bacterial proteins or selenium-containing enzymes. In existing research, the conversion rate of organic selenium in this bacterium has reached over 90%, and under optimized conditions, it can even reach over 97%.

[0003] In the selenium-enriched fermentation of *Rhodopseudomonas palustris*, the fermentation broth is also rich in extracellular polysaccharides. Existing technologies require alcohol precipitation to extract extracellular polysaccharides from the fermentation broth. For example, Chinese invention patent application No. 2020101872366, "Extracellular Polysaccharides of *Rhodopseudomonas palustris* and Their Preparation Method and Application," involves centrifuging the fermentation broth to obtain the supernatant, removing suspended solids from the supernatant using membrane filtration, followed by alcohol precipitation, centrifugation, collection of the precipitate, and freeze-drying to obtain crude polysaccharides.

[0004] Alcohol precipitation is a classic method for polysaccharide separation and purification, and a commonly used process for polysaccharide extraction. Its core principle is to utilize the characteristic that the solubility of polysaccharides in ethanol decreases sharply to achieve precipitation separation, usually requiring a final concentration of 80% to 90%. This process consumes a large amount of ethanol, and ethanol is a flammable, explosive, and volatile solvent. Its storage, use, and recycling processes pose significant safety hazards, requiring stringent explosion-proof requirements for production workshops, and greatly increasing the costs of plant construction and safe production operations.

[0005] Therefore, there is a need to develop a non-alcohol precipitation-dependent, green, and safe method for polysaccharide extraction, which is crucial for the development and utilization of extracellular polysaccharides from selenium-rich Rhodopseudomonas palustris. This invention aims to overcome the inherent defects of existing alcohol precipitation techniques and provide a completely new technical approach. Summary of the Invention

[0006] The purpose of this invention is to extract extracellular polysaccharides from the fermentation broth of selenium-rich Rhodopseudomonas marsh. The prepared extracellular polysaccharide dry powder is white in color, has high purity, and does not contain sodium selenite.

[0007] The present invention employs a method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas palustris* enriched with selenium. The *Rhodopseudomonas palustris* undergoes selenium-enriched fermentation to obtain a fermentation broth containing extracellular polysaccharides. Crucially, the above-mentioned method for extracting polysaccharides specifically includes: S1 fermentation broth pretreatment: The selenium-enriched Rhodopseudomonas sphagnum fermentation broth was centrifuged, filtered, and concentrated once to obtain a concentrated broth containing crude polysaccharides. S2 Decolorization: The primary concentrate was decolorized using a mixture of d-limonene and ethyl acetate under the action of aminated silica nanoparticles to obtain a colorless and transparent polysaccharide solution; wherein the volume ratio of the primary concentrate, d-limonene and ethyl acetate was 10:0.5~1.5:1.5~3. S3 Precipitation and Impurity Removal: Add an organic acid salt composed of magnesium citrate and ferrous malate to the above decolorized polysaccharide solution, stir and adjust the pH to 4.5-5.5, and centrifuge to remove the precipitate after the reaction; S4 Desalting and Purification: The solution obtained in step S3 is desalted and purified by dialysis or ultrafiltration to obtain a purified polysaccharide solution; S5 drying yields polysaccharide powder.

[0008] Furthermore, step S1 specifically involves: Take the fermentation broth of Rhodopseudomonas palustris after selenium enrichment fermentation, centrifuge at 14000r / min~16000r / min for 10min~20min to remove the bacterial cells, and take the supernatant; filter the supernatant through a membrane to obtain the filtrate; The filtrate was concentrated once to 1 / 10 to 1 / 5 of its original volume at 55℃~65℃ and a vacuum of 0.08MPa~0.1MPa to obtain a primary concentrate.

[0009] Furthermore, step S2 specifically involves: The primary concentrate, d-limonene, and ethyl acetate were mixed in a certain proportion. 0.4% to 0.6% (by weight of the primary concentrate) of aminated silica nanoparticles were added. The mixture was stirred at 30°C to 35°C and 100 to 200 rpm for 30 to 40 minutes, allowed to stand for 30 to 60 minutes, and then centrifuged at 4000 to 5000 rpm for 5 to 15 minutes. The lower aqueous phase was separated to obtain a colorless and transparent polysaccharide solution.

[0010] Specifically, the aforementioned aminated silica nanoparticles are prepared by dispersing silica particles with a particle size of 300 nm to 500 nm in anhydrous ethanol, ultrasonically treating them, and then sequentially adding an organic base catalyst and 3-aminopropyltriethoxysilane. The reaction is carried out under nitrogen protection at 55 °C to 65 °C for 2 h to 4 h. The mass ratio of the silica particles, 3-aminopropyltriethoxysilane, and organic base catalyst is 100:0.4 to 0.6:0.5 to 1.5. The organic base catalyst is 1,8-diazabicycloundec-7-ene and triethylamine in a mass ratio of 1:0.2 to 0.5.

[0011] Specifically, the S3 steps are as follows: Magnesium citrate was added to the decolorized polysaccharide solution to make the mass concentration of magnesium citrate in the solution 0.8% to 1.2%; after stirring and dissolving, ferrous malate was added; after stirring and dissolving, the pH value was adjusted to 4.5 to 5.5, and the solution was allowed to stand for 10 to 14 hours to form a precipitate in the polysaccharide solution. The solution was then centrifuged at 8000 to 12000 r / min for 10 to 15 minutes, and the supernatant was collected; the mass ratio of ferrous malate to magnesium citrate was 1:(1 to 1.5).

[0012] It should be noted that the dialysis method described above is specifically: Using a dialysis bag with a molecular weight cutoff of 3.5 kDa pretreated with EDTA solution, dialysis was performed in deionized water at 25℃~30℃. The volume of deionized water used in each dialysis was 50 to 60 times the volume of the supernatant obtained in step S3. The dialysis solution was changed 3 to 5 times until dialysis was completed, and a purified polysaccharide solution was obtained.

[0013] It should be noted that the ultrafiltration method described above is specifically as follows: An ultrafiltration membrane with a molecular weight cutoff of 3kDa to 5kDa was used to desalinate the supernatant obtained in step S3 under an operating pressure of 0.1MPa to 0.5MPa and a temperature of 25℃ to 30℃. During the ultrafiltration desalination process, deionized water was added until the conductivity no longer changed, resulting in a purified polysaccharide solution.

[0014] Preferably, the drying process described in step S5 is either freeze drying or spray drying.

[0015] Specifically, the above-mentioned freeze-drying is as follows: The polysaccharide was dried using an in-situ freeze dryer. The secondary concentrate was placed on a tray, and the cold trap temperature was set to -45℃ to -35℃, the vacuum degree to 9 Pa to 11 Pa, and the drying time to 22 h to 26 h to obtain the above-mentioned polysaccharide powder. The specific process of the secondary concentration is as follows: the purified polysaccharide solution obtained in step S4 is concentrated to 1 / 4 to 1 / 2 of its original volume at 55℃ to 65℃ and a vacuum degree of 0.08 MPa to 0.1 MPa to obtain the secondary concentrate. More specifically, the above-mentioned spray drying is as follows: The purified polysaccharide solution obtained in step S4 was pumped to a spray drying tower via a peristaltic pump and spray dried at a feed rate of 300 mL / h to 500 mL / h. The inlet temperature was controlled at 160℃ to 180℃, the outlet temperature at 75℃ to 85℃, and the hot air flow rate at 0.6 m³ / h. 3 / min~0.8m 3 The powder at the bottom of the drying tower is collected at a rate of 1000 m / min to obtain the polysaccharide dry powder described above.

[0016] Compared with the prior art, the present invention has the following advantages: This invention breaks through the traditional alcohol precipitation process essential for polysaccharide extraction, providing a novel polysaccharide extraction technology. Through the synergistic effect of three core steps—decolorization, precipitation for impurity removal, and desalting purification—this invention achieves high-purity, high-yield polysaccharide extraction. Specifically, the decolorization process employs a mixed solvent system of d-limonene and ethyl acetate, selectively dissolving and removing fat-soluble pigments through polar gradient distribution. Simultaneously, aminated silica nanoparticles are used, utilizing the electrostatic attraction and specific adsorption between their surface amine groups and pigment molecules to significantly improve decolorization efficiency and greatly reduce polysaccharide loss. The precipitation for impurity removal innovatively employs a magnesium citrate and ferrous malate composite salt system, efficiently flocculating and removing impurities such as proteins under specific pH conditions, further purifying the polysaccharide solution. The desalting purification step thoroughly removes small molecule impurities and inorganic ions through dialysis or ultrafiltration, thus ensuring the high purity of the final product.

[0017] Compared to traditional alcohol precipitation, which not only fails to effectively break down the pigment-inorganic selenium chelate, resulting in high levels of residual inorganic selenium in the product and posing safety hazards, but also requires the use of large quantities of flammable and explosive ethanol, imposing stringent safety requirements, this invention utilizes a d-limonene-ethyl acetate mixed solvent to efficiently decolorize while simultaneously disrupting the chelate structure between the pigment and inorganic selenium, releasing Se. 4+ It is completely removed during subsequent dialysis or ultrafiltration, resulting in inorganic selenium residues below the detection limit, which greatly improves the food safety of the product.

[0018] Furthermore, this invention avoids the use of high-concentration ethanol throughout the process, resulting in extremely low levels of organic solvent residue in the product, or even no detectable organic solvent residue. Some samples showed only trace amounts of d-limonene and ethyl acetate, which are far below food safety limits and comply with food additive standards, significantly improving the safety and compliance of the product.

[0019] In summary, this invention achieves multiple beneficial effects for the first time in the field of polysaccharide extraction from selenium-enriched Rhodopseudomonas palustris fermentation broth: zero alcohol precipitation, zero inorganic selenium residue, high polysaccharide yield, and high product purity. It provides reliable and irreplaceable technical support for the extraction and development of polysaccharides from selenium-enriched Rhodopseudomonas palustris fermentation broth. Attached Figure Description

[0020] Figure 1 This is the infrared spectrum of sample 1 of the polysaccharide dry powder of the present invention.

[0021] Figure 2 This is a high-performance liquid chromatogram of the monosaccharide composition of sample 1 of the polysaccharide dry powder of the present invention.

[0022] Figure 3 This is the standard curve plotted for the inorganic selenium residue test of this invention. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Unless otherwise specified in the examples, the procedures can be followed according to conventional conditions; unless the manufacturers of the reagents or instruments used are specified, they are all conventional products that can be purchased commercially.

[0029] The Rhodopseudomonas palustris used in this invention was purchased from Zhili Zhongte (Wuhan) Biotechnology Co., Ltd., product number bio-52488.

[0030] Example 1 The specific process for preparing the aminated silica nanoparticles used for decolorization in this embodiment is as follows: 400nm silica particles were pre-treated by drying at 60℃ under vacuum for 2 hours. Add the dried silica particles to anhydrous ethanol, using 1L of anhydrous ethanol for every 100g of silica particles, to ensure that the silica particles are fully dispersed. The particles were ultrasonically treated at 300W power and 30℃ for 30 minutes to break up the agglomeration and form a uniform suspension. Under nitrogen protection, 1,8-diazabicycloundec-7-ene and triethylamine were added dropwise in sequence. After the addition was completed, the mixture was stirred for 10 min. 3-aminopropyltriethoxysilane was added dropwise at a rate of 1 mL / min. The mixture was reacted at 60 °C for 3 h under nitrogen protection to prepare aminated silica nanoparticles, which were designated as SiO2 sample 1. In this embodiment, the mass ratio of silica particles, 3-aminopropyltriethoxysilane, and organic base catalyst is 100:0.5:1.0, and the mass ratio of 1,8-diazabicycloundec-7-ene and triethylamine in the organic base catalyst is 1:0.3.

[0031] Example 2 The specific process for preparing the aminated silica nanoparticles used for decolorization in this embodiment is as follows: Silica particles with a particle size of 300 nm were pre-treated by drying at 65°C under vacuum for 1.5 h. Add the dried silica particles to anhydrous ethanol, using 0.8L of anhydrous ethanol for every 100g of silica particles, to ensure that the silica particles are fully dispersed. The particles were ultrasonically treated at 400W power and 28℃ for 25 minutes to break up the agglomeration and form a uniform suspension. Under nitrogen protection, 1,8-diazabicycloundec-7-ene and triethylamine were added dropwise in sequence. After the addition was completed, the mixture was stirred for 12 min. 3-aminopropyltriethoxysilane was added dropwise at a rate of 1.2 mL / min. The mixture was reacted at 55 °C for 4 h under nitrogen protection to prepare aminated silica nanoparticles, which were designated as SiO2 sample 2. In this embodiment, the mass ratio of silica particles, 3-aminopropyltriethoxysilane, and organic base catalyst is 100:0.4:1.5, and the mass ratio of 1,8-diazabicycloundec-7-ene and triethylamine in the organic base catalyst is 1:0.5.

[0032] Example 3 The specific process for preparing the aminated silica nanoparticles used for decolorization in this embodiment is as follows: Silica particles with a particle size of 500 nm were pre-treated by drying at 55°C under vacuum for 3 hours. Add the dried silica particles to anhydrous ethanol, using 1L of anhydrous ethanol for every 100g of silica particles, to ensure that the silica particles are fully dispersed. The particles were ultrasonically treated at 200W power and 32℃ for 35 minutes to break up the agglomeration and form a uniform suspension. Under nitrogen protection, 1,8-diazabicycloundec-7-ene and triethylamine were added dropwise in sequence. After the addition was completed, the mixture was stirred for 10 min. 3-aminopropyltriethoxysilane was added dropwise at a rate of 0.8 mL / min. The mixture was reacted at 65 °C for 2 h under nitrogen protection to prepare aminated silica nanoparticles, which were designated as SiO2 sample 3. In this embodiment, the mass ratio of silica particles, 3-aminopropyltriethoxysilane, and organic base catalyst is 100:0.6:0.5, and the mass ratio of 1,8-diazabicycloundec-7-ene and triethylamine in the organic base catalyst is 1:0.2.

[0033] Example 4 In this embodiment, the preparation and fermentation of the Rhodopseudomonas palustris seed culture used for fermentation are carried out. The specific process is as follows: Step 1: Activation of Rhodopseudomonas palustris freeze-dried powder: Add 0.5 mL of liquid seed culture medium to a freeze-dried tube of Rhodopseudomonas palustris to dissolve the bacterial powder. Transfer the dissolved bacterial solution to the liquid seed culture medium and mix well to obtain a bacterial suspension. Spread 0.2 mL of bacterial suspension onto a solid seed culture medium and place it in an anaerobic incubator for 5 days at a temperature of 30°C, with a 40W incandescent lamp (20 cm away) and a light intensity of 4000 Lux. The liquid seed culture medium consists of 1.0 g / L sodium acetate, 1.0 g / L ammonium chloride, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium chloride, 1.0 g / L sodium chloride, 0.5 g / L calcium chloride, water, and pH 6.8. The solid seed culture medium consists of 1.0 g / L sodium acetate, 1.0 g / L ammonium chloride, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium chloride, 1.0 g / L sodium chloride, 0.5 g / L calcium chloride, 1.5 g / L agar, water, and pH 6.8.

[0034] Step 2: Preparation of Rhodopseudomonas palustris seed culture: Pick a single colony from a plate and inoculate it into 100 mL of liquid seed culture medium. Place it in an anaerobic incubator and incubate at 30℃ with a 40W incandescent lamp (20 cm away) and a light intensity of 4000 Lux. Incubate until the logarithmic phase to obtain Rhodopseudomonas palustris seed solution, which is denoted as seed solution. The seed solution can be stored at 4℃ in the dark for 7 days.

[0035] Step 3: Selenium-enriched fermentation of Rhodopseudomonas palustris: The seed culture was inoculated into the fermentation medium at 5% by volume. The culture was statically incubated for 7 days at 28℃ under a 40W incandescent lamp (20cm away) with a light intensity of 4000 Lux. Sodium selenite was added on days 2 to 5 of the fermentation culture. Specifically, the amount of sodium selenite added was 10 mg / L on day 2, 20 mg / L on day 3, 20 mg / L on day 4, and 10 mg / L on day 5. The cumulative concentration of sodium selenite reached 60 mg / L on day 5. After 7 days of culture, the fermentation was terminated, and the fermentation broth was obtained for later use. The fermentation medium contains 1.5 g / L sodium acetate, 2.0 g / L yeast extract, 1.0 g / L sodium chloride, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium chloride, 0.1 g / L calcium chloride, 0.5 mg / L ferrous chloride, 0.5 mg / L manganese chloride, 0.5 mg / L boric acid, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, and 100 mg / L inositol, with a pH of 6.8.

[0036] Example 5 This embodiment describes the extraction of extracellular polysaccharides from selenium-enriched fermentation broth. The specific steps are as follows: S1 fermentation broth pretreatment: Take the fermentation broth, centrifuge at 15000 r / min for 15 min to remove the cells, and take the supernatant. Filter the supernatant through a PES membrane with a pore size of 0.22 μm at a flow rate of 100 mL / min to obtain the filtrate. Concentrate the filtrate once at 60℃ and a vacuum of 0.09 MPa to reduce the volume to 1 / 7 of the original volume to obtain the concentrated solution.

[0037] S2 decolorization: The primary concentrate, d-limonene, and ethyl acetate were mixed at a volume ratio of 10:0.8:2.2. 0.5% of the mass of the primary concentrate, SiO2 sample 1, was added. The mixture was stirred at 32℃ and 150 r / min for 35 min, allowed to stand for 45 min, and centrifuged at 4500 r / min for 10 min. The lower aqueous phase was separated to obtain a colorless and transparent polysaccharide solution after decolorization, which was designated as decolorized polysaccharide solution sample 1.

[0038] S3 precipitation for impurity removal: Magnesium citrate was added to the decolorized polysaccharide solution obtained in step S2 to make the mass concentration of magnesium citrate in the solution 0.9%; After stirring and dissolving, ferrous malate is added, with a mass ratio of ferrous malate to magnesium citrate of 1:1.2. After stirring and dissolving again, adjust the pH to 4.9, let stand for 12 hours, and a precipitate will form in the polysaccharide solution. Centrifuge at 9000 r / min for 12 min and collect the supernatant.

[0039] S4 desalting and purification: Use dialysis bags with a molecular weight cutoff of 3.5 kDa that have been pretreated with EDTA solution; Dialysis was performed in deionized water at 28°C, and the volume of deionized water used in each dialysis was 55 times the volume of the supernatant obtained in step S3. The dialysis solution was changed 4 times until dialysis was completed, and the purified polysaccharide solution was obtained, which was recorded as purified polysaccharide solution sample 1.

[0040] S5 drying: The purified polysaccharide solution obtained in step S4 was concentrated to 1 / 3 of its original volume at 60°C and a vacuum of 0.09 MPa to obtain a secondary concentrate. The polysaccharide was dried using an in-situ freeze dryer. The resulting secondary concentrate was placed on a tray, and the cold trap temperature was set to -40℃, the vacuum degree to 10Pa, and the drying time to 24h to obtain polysaccharide dry powder, which was designated as polysaccharide sample 1.

[0041] Example 6 This embodiment describes the extraction of extracellular polysaccharides from selenium-enriched fermentation broth. The specific steps are as follows: S1 fermentation broth pretreatment: Take the fermentation broth and centrifuge it at 14000 r / min for 20 min to remove the cells. Take the supernatant. Filter the supernatant through a PES membrane with a pore size of 0.22 μm at a flow rate of 120 mL / min to obtain the filtrate. Concentrate the filtrate once at 55℃ and a vacuum of 0.1 MPa to reduce the volume to 1 / 5 of the original volume to obtain the concentrated solution.

[0042] S2 decolorization: The primary concentrate, d-limonene, and ethyl acetate were mixed at a volume ratio of 10:0.5:3. 0.6% of the primary concentrate by mass of SiO2 sample 2 was added. The mixture was stirred at 35°C and 200 r / min for 30 min, allowed to stand for 60 min, and centrifuged at 5000 r / min for 5 min. The lower aqueous phase was separated to obtain a decolorized colorless and transparent polysaccharide solution, which was designated as decolorized polysaccharide solution sample 2.

[0043] S3 precipitation for impurity removal: Magnesium citrate was added to the decolorized polysaccharide solution obtained in step S2 to make the mass concentration of magnesium citrate in the solution 0.8%; After stirring and dissolving, add ferrous malate, with a mass ratio of ferrous malate to magnesium citrate of 1:1.5. After stirring and dissolving again, adjust the pH to 4.5, let stand for 10 hours, and a precipitate will form in the polysaccharide solution. Centrifuge at 12000 r / min for 10 min and collect the supernatant.

[0044] S4 desalting and purification: Use dialysis bags with a molecular weight cutoff of 3.5 kDa that have been pretreated with EDTA solution; Dialysis was performed in deionized water at 25°C, and the volume of deionized water used in each dialysis was 60 times the volume of the supernatant obtained in step S3. The dialysis solution was changed 3 times until dialysis was completed, and the purified polysaccharide solution was obtained, which was recorded as purified polysaccharide solution sample 2.

[0045] S5 drying: The purified polysaccharide solution obtained in step S4 was concentrated to half of its original volume at 55°C and a vacuum of 0.1 MPa to obtain a secondary concentrate. The polysaccharide was dried using an in-situ freeze dryer. The resulting secondary concentrate was placed on a tray, and the cold trap temperature was set to -45℃, the vacuum degree to 9Pa, and the drying time to 26h to obtain polysaccharide dry powder, which was designated as polysaccharide sample 2.

[0046] Example 7 This embodiment describes the extraction of extracellular polysaccharides from selenium-enriched fermentation broth. The specific steps are as follows: S1 fermentation broth pretreatment: Take the fermentation broth, centrifuge at 16000 r / min for 10 min to remove the cells, and take the supernatant. Filter the supernatant through a PES membrane with a pore size of 0.22 μm at a flow rate of 80 mL / min to obtain the filtrate. Concentrate the filtrate once at 65℃ and a vacuum of 0.08 MPa to reduce the volume to 1 / 10 of the original volume to obtain the concentrated solution.

[0047] S2 decolorization: The primary concentrate, d-limonene, and ethyl acetate were mixed at a volume ratio of 10:1.5:1.5. 0.4% (by mass) of SiO2 sample 3 of the primary concentrate was added. The mixture was stirred at 30°C and 100 r / min for 40 min, allowed to stand for 30 min, and centrifuged at 4000 r / min for 15 min. The lower aqueous phase was separated to obtain a decolorized, colorless, and transparent polysaccharide solution, which was designated as decolorized polysaccharide solution sample 3.

[0048] S3 precipitation for impurity removal: Magnesium citrate was added to the decolorized polysaccharide solution obtained in step S2 to make the mass concentration of magnesium citrate in the solution 1.2%; After stirring and dissolving, add ferrous malate, with a mass ratio of ferrous malate to magnesium citrate of 1:1. After stirring and dissolving again, adjust the pH to 5.5, let stand for 14 hours, and a precipitate will form in the polysaccharide solution. Centrifuge at 8000 r / min for 15 min and collect the supernatant.

[0049] S4 desalting and purification: Use dialysis bags with a molecular weight cutoff of 3.5 kDa that have been pretreated with EDTA solution; Dialysis was performed in deionized water at 30°C, and the volume of deionized water used in each dialysis was 50 times the volume of the supernatant obtained in step S3. The dialysis fluid was changed 5 times until dialysis was completed, and the purified polysaccharide solution was obtained, which was recorded as purified polysaccharide solution sample 3.

[0050] S5 drying: The purified polysaccharide solution obtained in step S4 was pumped to a spray drying tower via a peristaltic pump and spray dried at a feed rate of 400 mL / h, with the inlet temperature controlled at 160℃, the outlet temperature at 80℃, and the hot air flow rate at 0.7 m³ / h. 3 The powder at the bottom of the drying tower was collected at a speed of 1000 min to obtain polysaccharide dry powder, which was designated as polysaccharide sample 3.

[0051] Example 8 This embodiment describes the extraction of extracellular polysaccharides from selenium-enriched fermentation broth. The specific steps are as follows: S1 fermentation broth pretreatment: Same as step S1 in Example 5.

[0052] S2 Decolorization: Same as step S2 in Example 5.

[0053] S3 Precipitation and Impurity Removal: Same as step S3 in Example 5.

[0054] S4 desalting and purification: An ultrafiltration membrane with a molecular weight cutoff of 4 kDa was used to desalinate the supernatant obtained in step S3 under an operating pressure of 0.3 MPa and a temperature of 28°C. Deionized water was added during the ultrafiltration desalination process until the conductivity no longer changed, resulting in a purified polysaccharide solution, which was designated as purified polysaccharide solution sample 4.

[0055] S5 drying: The purified polysaccharide solution obtained in step S4 was pumped to a spray drying tower via a peristaltic pump and spray dried at a feed rate of 500 mL / h, with the inlet temperature controlled at 180℃, the outlet temperature at 85℃, and the hot air flow rate at 0.6 m³ / h. 3 The powder at the bottom of the drying tower was collected at a speed of 1000 min to obtain polysaccharide dry powder, which was recorded as polysaccharide sample 4.

[0056] Example 9 This embodiment describes the extraction of extracellular polysaccharides from selenium-enriched fermentation broth. The specific steps are as follows: S1 fermentation broth pretreatment: Same as step S1 in Example 5.

[0057] S2 Decolorization: Same as step S2 in Example 5.

[0058] S3 Precipitation and Impurity Removal: Same as step S3 in Example 5.

[0059] S4 desalting and purification: An ultrafiltration membrane with a molecular weight cutoff of 3 kDa was used to desalinate the supernatant obtained in step S3 under an operating pressure of 0.5 MPa and a temperature of 25°C. Deionized water was added during the ultrafiltration desalination process until the conductivity no longer changed, resulting in a purified polysaccharide solution, which was designated as purified polysaccharide solution sample 5.

[0060] S5 drying: The purified polysaccharide solution obtained in step S4 was pumped to a spray drying tower via a peristaltic pump and spray dried at a feed rate of 300 mL / h, with the inlet temperature controlled at 160℃, the outlet temperature at 75℃, and the hot air flow rate at 0.8 m³ / h. 3 The powder at the bottom of the drying tower was collected at a speed of 1000 min to obtain polysaccharide dry powder, which was recorded as polysaccharide sample 5.

[0061] Example 10 This embodiment describes the extraction of extracellular polysaccharides from selenium-enriched fermentation broth. The specific steps are as follows: S1 fermentation broth pretreatment: Same as step S1 in Example 5.

[0062] S2 Decolorization: Same as step S2 in Example 5.

[0063] S3 Precipitation and Impurity Removal: Same as step S3 in Example 5.

[0064] S4 desalting and purification: An ultrafiltration membrane with a molecular weight cutoff of 5 kDa was used to desalinate the supernatant obtained in step S3 under an operating pressure of 0.1 MPa and a temperature of 30°C. Deionized water was added during the ultrafiltration desalination process until the conductivity no longer changed, resulting in a purified polysaccharide solution, which was designated as purified polysaccharide solution sample 6.

[0065] S5 drying: The purified polysaccharide solution obtained in step S4 was concentrated to 1 / 4 of its original volume at 65°C and a vacuum of 0.08 MPa to obtain a secondary concentrate. The polysaccharide was dried using an in-situ freeze dryer. The resulting secondary concentrate was placed on a tray, and the cold trap temperature was set to -35℃, the vacuum degree to 11Pa, and the drying time to 22h to obtain polysaccharide powder, which was designated as polysaccharide sample 6.

[0066] Comparative Example 1 In this comparative example, extracellular polysaccharides were extracted from the selenium-enriched fermentation broth. The specific steps were the same as in Example 5, except that in step S2, SiO2 sample 1 was not used. Instead, 0.5% of commercially available silica particles with a particle size of 400 nm were added by mass of the concentrate. After decolorization, decolorized polysaccharide solution control 1 was obtained. The subsequent steps were the same as in Example 5, and purified polysaccharide solutions were obtained sequentially, which were designated as purified polysaccharide solution control 1. Extracellular polysaccharide dry powder was prepared and designated as polysaccharide control 1.

[0067] Comparative Example 2 In this comparative example, extracellular polysaccharides were extracted from the selenium-enriched fermentation broth. The specific steps were the same as in Example 5, except that step S2 did not use d-limonene and SiO2 sample 1. The specific steps were as follows: S1 fermentation broth pretreatment: Same as step S1 in Example 5.

[0068] S2 decolorization: The primary concentrate and ethyl acetate were mixed at a volume ratio of 10:3. Activated carbon of 0.5% by mass of the primary concentrate was added. The mixture was stirred at 32°C and 150 r / min for 35 min. After standing and separating into layers, the lower aqueous phase solution was retained to obtain the decolorized polysaccharide solution, which was designated as decolorized polysaccharide solution reference standard 2.

[0069] S3 Precipitation and Impurity Removal: Same as step S3 in Example 5.

[0070] S4 Desalting and Purification: Same as step S4 in Example 5, to obtain a purified polysaccharide solution, which is designated as purified polysaccharide solution control 2.

[0071] S5 Drying: The polysaccharide dry powder was obtained by the same step as in Example 5, and was designated as polysaccharide control 2.

[0072] Comparative Example 3 In this comparative example, extracellular polysaccharides were extracted from the selenium-enriched fermentation broth. The difference from Example 5 is that step S3 was omitted. The specific steps are as follows: S1 fermentation broth pretreatment: Same as step S1 in Example 5.

[0073] S2 Decolorization: Same as step S2 in Example 5.

[0074] S3 Desalting and Purification: Same as step S4 in Example 5, to obtain a purified polysaccharide solution, which is designated as purified polysaccharide solution control standard 3.

[0075] S4 drying: The purified polysaccharide solution obtained in step S3 was concentrated to 1 / 3 of its original volume at 60°C and a vacuum of 0.09 MPa to obtain a secondary concentrate. The secondary concentrate was dried using an in-situ freeze dryer. The secondary concentrate was placed on a tray and freeze-dried according to the parameters set in step S5 of Example 5 to obtain polysaccharide powder, which was designated as polysaccharide reference standard 3.

[0076] Comparative Example 4 In this comparative example, extracellular polysaccharides were extracted from the selenium-enriched fermentation broth. The difference from Example 5 is that step S4 was omitted. The specific steps are as follows: S1 fermentation broth pretreatment: Same as step S1 in Example 5.

[0077] S2 Decolorization: Same as step S2 in Example 5.

[0078] S3 Precipitation and Impurity Removal: Same as step S3 in Example 5, but the supernatant is recorded as the purified polysaccharide solution reference standard 4.

[0079] S4 drying: The supernatant obtained in step S3 was concentrated to 1 / 3 of its original volume at 60°C and a vacuum of 0.09 MPa to obtain a secondary concentrate. The secondary concentrate was dried using an in-situ freeze dryer. The secondary concentrate was placed on a tray and freeze-dried according to the parameters set in step S5 of Example 5 to obtain polysaccharide dry powder, which was designated as polysaccharide reference standard 4.

[0080] Comparative Example 5 In this comparative example, extracellular polysaccharides were extracted from the selenium-enriched fermentation broth. The specific steps were the same as in Example 5, except that ferrous malate was not used in step S3, only magnesium citrate was used, and the mass concentration of magnesium citrate in the solution was 1.5%. The subsequent steps were the same as in Example 5, and purified polysaccharide solutions were obtained sequentially, which were designated as purified polysaccharide solution control 5. Extracellular polysaccharide dry powder was prepared, which was designated as polysaccharide control 5.

[0081] Comparative Example 6 In this comparative example, extracellular polysaccharides were extracted from the selenium-enriched fermentation broth. The specific steps were the same as in Example 5, except that in step S3, ferrous malate and magnesium citrate were not used, but FeSO4 was used instead. The amount of FeSO4 used was 1.5% of the solution mass. The subsequent steps were the same as in Example 5, and purified polysaccharide solutions were obtained sequentially, which were designated as purified polysaccharide solution control 6. Extracellular polysaccharide dry powder was prepared, which was designated as polysaccharide control 6.

[0082] Comparative Example 7 In this comparative example, extracellular polysaccharides were extracted from the selenium-enriched fermentation broth using the traditional alcohol precipitation method. The specific steps were as follows: S1 fermentation broth pretreatment: Same as step S1 in Example 5.

[0083] S2 alcohol precipitation method for polysaccharide extraction: The concentrate was added to 9 times its volume of anhydrous ethanol to make the alcohol precipitation concentration 90%, and the precipitate was precipitated at 4°C for 12 h; centrifuged at 5000 r / min for 20 min and the precipitate was collected; the precipitate was washed with anhydrous ethanol and acetone respectively.

[0084] S3 Drying: Same as step S5 in Example 5, to obtain extracellular polysaccharide dry powder, which is designated as polysaccharide control 7.

[0085] Table 1 summarizes the sample names involved in the embodiments and comparative examples of the present invention.

[0086] Table 1: Summary of Sample Names Involved in Examples and Comparative Examples Analysis and Testing I. Infrared Spectroscopic Analysis of Polysaccharide Samples Weigh 1.0 mg of polysaccharide sample 1 and 200 mg of potassium bromide crystals, mix them in a mortar, dry them under an infrared lamp for 15 min, and grind and mix them thoroughly. Press them into a semi-transparent thin sheet using a special tablet press for infrared spectrometers, and place them on an infrared spectrometer for detection in the wavelength range of 400–4000 cm⁻¹. -1 The results are shown Figure 1 .

[0087] Depend on Figure 1 The results show that polysaccharide sample 1 has a characteristic absorption peak for polysaccharides: 3404.72 cm⁻¹. -1 A strong and broad absorption peak exists at 2930.23 cm⁻¹, which is the strong absorption peak of the OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The nearby moderate-intensity peak is the absorption peak of the CH stretching vibration of methyl (-CH3) or methine (-CH2); 1648.42 cm⁻¹ -1The absorption peaks are due to the C=O stretching vibration of the carbonyl group or the hydration vibration of polysaccharides; 1450-1200 cm⁻¹ -1 The absorption peak is at 1412.18 cm⁻¹. -1 1374.37cm -1 1251.30cm -1 These are absorption peaks from the angular vibrations of CH4, which, along with the stretching vibrations of CH4, constitute the characteristic absorption of the sugar ring; 1049.94 cm⁻¹ -1 This represents the bending vibration of the CO bond in the COH or COC structure; 916.78 cm⁻¹ -1 The peak represents the asymmetric ring stretching vibration of the pyran ring; 890.06 cm⁻¹ -1 The region is a characteristic region of β-pyranoside bond, suggesting that the polysaccharide contains β-pyranose.

[0088] II. Analysis of total sugar content and monosaccharide composition of the extracted polysaccharides The total sugar content (mass ratio) in polysaccharide samples and reference standards was determined using the phenol-sulfuric acid method, and the results are shown in Table 2. Polysaccharide sample 1 was acid-hydrolyzed, and its monosaccharide composition was analyzed by high-performance liquid chromatography (HPLC), and the results are shown in Table 2. Figure 2 And Table 3.

[0089] Table 2: Total sugar content in polysaccharide samples and control standards Table 3: Monosaccharide components and percentage of total sugars in polysaccharide sample 1 As shown in Table 2, the present invention has a significant advantage in extraction purity. The total sugar content of the samples from the present invention remained stable between 91.5% and 92.9%, which directly proves that the three-step synergistic process of decolorization, impurity removal, and desalting of the present invention has stable and efficient purification capabilities, effectively removing non-sugar impurities such as proteins, pigments, and inorganic salts to obtain high-purity polysaccharide products. Furthermore, as shown in Table 3, the main components of the polysaccharide prepared by the present invention are mannose and glucose, containing only trace amounts of glucosamine, and mannose accounts for more than 70% of the total sugar content. Therefore, the product of the present invention is a polysaccharide product mainly composed of mannose.

[0090] Due to insufficient decolorization selectivity, the total sugar content of polysaccharide reference standards 1 and 2 decreased to 85.2% and 81.6%, respectively, indicating that their processes did not thoroughly remove impurities. The total sugar content of polysaccharide reference standard 3 decreased significantly to 76.9%, proving that step S3 is crucial for removing large non-sugar impurities such as proteins and colloids; its absence leads to a significant drop in the total sugar content of the product. Polysaccharide reference standard 4 had the lowest total sugar content among all samples, demonstrating that omitting step S4 results in a large amount of inorganic salt contamination in the final product. Step S4 is essential for obtaining high-purity, accurately measurable polysaccharide products. The total sugar content of polysaccharide reference standards 5 and 6 was still significantly lower than that of this invention, indicating that the specific combination of magnesium citrate and ferrous malate has a key synergistic effect in achieving deep impurity removal and obtaining high-purity products. Polysaccharide reference standard 7 obtained by the traditional alcohol precipitation method had a total sugar content of 86.5%, reflecting the limitations of alcohol precipitation as a non-selective precipitation process. It is difficult to effectively separate polysaccharides from coexisting precipitable impurities, naturally limiting product purity.

[0091] III. Verification of Decolorization Effect Decolorized polysaccharide solution samples 1-3 and decolorized polysaccharide solution standards 1 and 2 were taken respectively. The transmittance at a wavelength of 800 nm was measured by spectrophotometer and the transmittance was calculated. Purified water was used as a control. The results are shown in Table 4.

[0092] Table 4: Verification Results of Decolorization Effect As shown in Table 4, the decolorization of the polysaccharide solution obtained by the present invention using the d-limonene and ethyl acetate-aminated SiO2 system is colorless and transparent, with a transmittance of more than 95%, demonstrating the excellent and stable decolorization effect of the present invention.

[0093] Comparative Example 1 used unaminated ordinary silica, which showed a significant decrease in light transmittance, indicating that amination modification is crucial for targeted pigment adsorption and improved decolorization efficiency. Comparative Example 2 used the traditional ethyl acetate-activated carbon method, which had the worst decolorization effect, and activated carbon also had an adsorption effect on polysaccharides.

[0094] IV. Testing of Polysaccharide Extraction Efficiency Purified polysaccharide solution samples 1-6 and purified polysaccharide solution reference standards 1-6 were used as samples after polysaccharide extraction. To ensure the scientific validity and consistency of the comparison, the starting material variables before extraction were controlled, and the concentrate from Example 5 was used as the sample before polysaccharide extraction.

[0095] The total sugar content before and after polysaccharide extraction was determined by the phenol-sulfuric acid method, and the polysaccharide yield was calculated. The results are shown in Table 5.

[0096] Polysaccharide yield = C 提取后 / C提取前 ×100%, where C 提取后 C represents the total sugar content in the extracted polysaccharide sample. 提取前 This indicates the total sugar content in the polysaccharide sample before extraction.

[0097] Table 5: Test results of polysaccharide extraction efficiency As shown in Table 5, the polysaccharide yield of each sample in this invention remained above 85%, proving that the process route of this invention can retain the target polysaccharide to the maximum extent while achieving efficient purification.

[0098] Comparative Examples 1 and 2 suffered from low decolorization efficiency and non-specific adsorption, resulting in polysaccharide loss and significantly reduced yields. Comparative Example 3 omitted the S3 purification step; although its polysaccharide yield was not low, the resulting polysaccharide powder had poor purity and high inorganic selenium residue, demonstrating that the S3 step is indispensable for obtaining high-purity and safe products.

[0099] Comparative Example 4, which omits the S4 desalting step, exhibits the lowest polysaccharide yield. As indicated by its extremely low total sugar content, the product is essentially a mixture of polysaccharides and a large amount of inorganic salts. This demonstrates the necessity of the S4 step; omitting it results in a low-purity product that cannot be accurately measured.

[0100] In Comparative Example 5, only magnesium citrate was used, and in Comparative Example 6, FeSO4 precipitation was used for impurity removal. The polysaccharide yields of both examples were lower than those of the present invention, and their total sugar content was also correspondingly lower. This demonstrates that the specific combination of magnesium citrate and ferrous malate of the present invention has a synergistic effect on impurity removal and on improving polysaccharide yield and purity.

[0101] V. Inorganic Selenium Residue Testing o-Phenylenediamine can undergo a complexation reaction with selenite in acidic media to form a complex. This complex can be extracted by toluene at pH 2 and has a maximum absorption peak at a wavelength of 334 nm.

[0102] Accurately weigh 2.19g of sodium selenite, dissolve it in deionized water, add it to a 1000mL volumetric flask, and make up to volume to prepare a 1000μg / mL selenium standard solution. When needed, take 1mL and make up to 1000mL to prepare a μg / mL selenium standard solution. Take 0, 5, 10, 15, 20, and 25 mL of 1 μg / mL selenium standard solution into beakers, add water, and then add 0.2 mL of 4% NaF solution, 2 mL of 0.1 mol / L EDTA-Na2 solution, and 3 mL of o-phenylenediamine solution, respectively. Adjust the pH to 2.0, react at 45℃ for 50 min, transfer to a separatory funnel, add toluene for extraction, shake, allow to stand for separation, discard the lower aqueous layer, transfer the toluene fraction to a quartz cuvette, and measure the absorbance at 334 nm. Plot a standard curve. The results are shown in [Figure number missing]. Figure 3 Where y = 0.0192x + 0.0064, R 2 =0.9998.

[0103] The absorbance values ​​of polysaccharide samples 1-6 and polysaccharide reference standards 1-7 were determined, and the inorganic selenium residue was calculated by substituting the values ​​into the selenium standard curve. The results are shown in Table 6.

[0104] Table 6: Test Results of Inorganic Selenium Residue As shown in Table 6, no inorganic selenium residue was detected in any of the polysaccharide samples of this invention, which further proves the thoroughness and effectiveness of the combination of d-limonene and ethyl acetate decolorization and chelate removal with subsequent purification steps for deep removal of inorganic selenium.

[0105] The inorganic selenium residues in all comparative examples were significantly higher than those in this invention. In particular, the residual amounts of inorganic selenium in polysaccharide references 3 and 4 were extremely high, demonstrating that S3 impurity removal and S4 desalination are two key steps in deep selenium removal. Notably, while the inorganic selenium residue in polysaccharide reference 5 was lower than in most comparative examples, it was still significantly present; and the residual amount in polysaccharide reference 6 was even higher. This indicates that the specific combination of ferrous malate and magnesium citrate is crucial for the complete removal of inorganic selenium. This is because, at the system pH, the organometallic complex formed by ferrous malate can more effectively target and remove free or chelated selenite ions through adsorption, co-precipitation, or coordination, creating a highly efficient synergistic effect with the flocculation and impurity removal function of magnesium citrate. The relatively high amount of inorganic selenium residue in polysaccharide reference 7 also demonstrates the inherent limitations of traditional processes in this regard.

[0106] The high levels of inorganic selenium residue in the comparative data (especially Comparative Example 2, which did not use d-limonene, and Comparative Example 6, which used only inorganic salt precipitation) suggest a synergistic effect between the d-limonene-ethyl acetate mixed solvent system and the subsequent organic acid salt purification and deep desalting steps. This system may have disrupted the exchange of pigments and inorganic selenium (Se) in the fermentation broth. 4+ The stable chelate structure formed between the molecules allows the encapsulated inorganic selenium to be released and thus completely removed in subsequent dialysis or ultrafiltration.

[0107] VI. Organic Solvent Residue Testing The residual organic solvents in polysaccharide samples 1–6 and polysaccharide reference standards 1–7 were tested using high performance gas chromatography. The results are shown in Table 7.

[0108] Table 7: Results of Organic Solvent Residue Tests As shown in Table 7, only trace amounts of d-limonene and ethyl acetate were detected in all polysaccharide samples of this invention, and these amounts were far below the food safety limits (d-limonene and ethyl acetate are both food flavorings that are explicitly permitted in GB 2760-2014 Food Additives Standard). Solvents such as ethanol and acetone were not detected, proving that the process of this invention can effectively remove organic solvents during post-processing, resulting in high product safety. High concentrations of ethanol and acetone residues were detected in polysaccharide reference standard 7, indicating that the traditional alcohol precipitation method also carries a high risk of organic solvent residue.

[0109] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas palustris* enriched with selenium, wherein *Rhodopseudomonas palustris* is fermented with selenium to obtain a fermentation broth containing extracellular polysaccharides, characterized in that... The method for extracting polysaccharides specifically includes: S1 fermentation broth pretreatment: The selenium-enriched Rhodopseudomonas sphagnum fermentation broth was centrifuged, filtered, and concentrated once to obtain a concentrated broth containing crude polysaccharides. S2 Decolorization: The primary concentrate was decolorized using a mixture of d-limonene and ethyl acetate under the action of aminated silica nanoparticles to obtain a colorless and transparent polysaccharide solution; wherein the volume ratio of the primary concentrate, d-limonene, and ethyl acetate was 10:0.5-1.5:1.5-3. S3 Precipitation and Impurity Removal: Add an organic acid salt composed of magnesium citrate and ferrous malate to the decolorized polysaccharide solution, stir and adjust the pH to 4.5-5.5, and centrifuge to remove the precipitate after the reaction; S4 Desalting and Purification: The solution obtained in step S3 is desalted and purified by dialysis or ultrafiltration to obtain a purified polysaccharide solution; S5 drying yields polysaccharide powder.

2. The method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas palustris* according to claim 1, characterized in that, Step S1 is as follows: Take the fermentation broth of Rhodopseudomonas palustris after selenium enrichment fermentation, centrifuge at 14000r / min~16000r / min for 10min~20min to remove the bacterial cells, and take the supernatant; filter the supernatant through a membrane to obtain the filtrate; The filtrate was concentrated once to 1 / 10 to 1 / 5 of its original volume at 55℃~65℃ and a vacuum of 0.08MPa~0.1MPa to obtain a primary concentrate.

3. The method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas pulverans* according to claim 1, characterized in that, Step S2 is as follows: The primary concentrate, d-limonene, and ethyl acetate were mixed in a certain proportion. 0.4% to 0.6% (by weight of the primary concentrate) of aminated silica nanoparticles were added. The mixture was stirred at 30°C to 35°C and 100 to 200 rpm for 30 to 40 minutes, allowed to stand for 30 to 60 minutes, and then centrifuged at 4000 to 5000 rpm for 5 to 15 minutes. The lower aqueous phase was separated to obtain a colorless and transparent polysaccharide solution.

4. The method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas palustris* according to claim 1, characterized in that, The aminated silica nanoparticles are prepared by dispersing silica particles with a particle size of 300 nm to 500 nm in anhydrous ethanol, ultrasonically treating them, and then sequentially adding an organic base catalyst and 3-aminopropyltriethoxysilane. The reaction is carried out under nitrogen protection at 55 °C to 65 °C for 2 h to 4 h. The mass ratio of silica particles, 3-aminopropyltriethoxysilane and organic base catalyst is 100:0.4 to 0.6:0.5 to 1.

5. The organic base catalyst is 1,8-diazabicycloundec-7-ene and triethylamine in a mass ratio of 1:0.2 to 0.

5.

5. The method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas palustris* according to claim 1, characterized in that, The specific steps in S3 are as follows: Magnesium citrate was added to the decolorized polysaccharide solution to make the mass concentration of magnesium citrate in the solution 0.8% to 1.2%; after stirring and dissolving, ferrous malate was added; after stirring and dissolving, the pH value was adjusted to 4.5 to 5.5, and the solution was allowed to stand for 10 to 14 hours to form a precipitate in the polysaccharide solution. The solution was then centrifuged at 8000 to 12000 r / min for 10 to 15 minutes, and the supernatant was collected; the mass ratio of ferrous malate to magnesium citrate was 1:(1 to 1.5).

6. The method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas palustris* according to claim 1, characterized in that, The dialysis method described is specifically: Using a dialysis bag with a molecular weight cutoff of 3.5 kDa pretreated with EDTA solution, dialysis was performed in deionized water at 25℃~30℃. The volume of deionized water used in each dialysis was 50 to 60 times the volume of the supernatant obtained in step S3. The dialysis solution was changed 3 to 5 times until dialysis was completed, and a purified polysaccharide solution was obtained.

7. The method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas palustris* according to claim 1, characterized in that, The ultrafiltration method described above is specifically: An ultrafiltration membrane with a molecular weight cutoff of 3kDa to 5kDa was used to desalinate the supernatant obtained in step S3 under an operating pressure of 0.1MPa to 0.5MPa and a temperature of 25℃ to 30℃. During the ultrafiltration desalination process, deionized water was added until the conductivity no longer changed, resulting in a purified polysaccharide solution.

8. The method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas palustris* according to claim 1, characterized in that, The drying process described in step S5 is either freeze drying or spray drying.

9. A method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas palustris* according to claim 8, characterized in that, The freeze-drying specifically refers to: The polysaccharide powder was obtained by drying using an in-situ freeze dryer, placing the secondary concentrate on a tray, setting the cold trap temperature to -45℃ to -35℃, the vacuum degree to 9Pa to 11Pa, and the drying time to 22h to 26h. The specific process of the secondary concentration is as follows: The purified polysaccharide solution obtained in step S4 was concentrated to 1 / 4 to 1 / 2 of its original volume at 55℃~65℃ and a vacuum of 0.08MPa~0.1MPa to obtain a secondary concentrate.

10. A method for extracting polysaccharides from the fermentation broth of *Rhodopseudomonas pulverans* according to claim 8, characterized in that, The spray drying specifically refers to: The purified polysaccharide solution obtained in step S4 was pumped to a spray drying tower via a peristaltic pump and spray dried at a feed rate of 300 mL / h to 500 mL / h. The inlet temperature was controlled at 160℃ to 180℃, the outlet temperature at 75℃ to 85℃, and the hot air flow rate at 0.6 m³ / h. 3 / min~0.8m 3 The powder at the bottom of the drying tower is collected at a rate of 1000 m / min to obtain the polysaccharide dry powder.

Images