A method for resourceful treatment of activated sludge

Abstract

The present application relates to sewage biological technology field, disclose a kind of resource processing method of activated sludge.The resource processing method of activated sludge includes the following steps: washing activated sludge obtains sediment sludge;Sediment sludge is mixed with water to obtain suspension, suspension is digested, and first supernatant and sludge residue are obtained by separation;First supernatant is impurity-removed by calcium salt solution and alcohol solvent to obtain first precipitate;First precipitate is dissolved and separated to obtain second supernatant;Second supernatant is impurity-removed by alcohol solvent to obtain second precipitate;Second precipitate is dissolved and separated to obtain third precipitate;Third precipitate is dissolved and separated to obtain third supernatant, and third supernatant is impurity-removed by alcohol solvent to obtain fourth precipitate;Fourth precipitate is purified to obtain heparinoid polysaccharide.This method has good impurity-removing effect, high polysaccharide yield, and the sludge residue obtained by separation can be used for fermentation to produce methane, and the heparinoid polysaccharide obtained has similar blood clotting function and anticancer potential to commercially available heparin, and has high application value.

Description

Technical Field

[0001] This invention relates to the field of sludge resource utilization technology, and in particular to a method for the resource utilization treatment of activated sludge. Background Technology

[0002] Municipal sludge treatment plants produce large quantities of activated sludge, with production increasing annually. Activated sludge is rich in organic matter and various microorganisms. Organic matter can be degraded into inorganic substances by these microorganisms, releasing chemical energy. Therefore, activated sludge can be considered a resource rich in chemical energy and substances. Activated sludge resource recovery can reduce energy consumption, decrease environmental pollution, and improve resource utilization efficiency, thus having significant environmental, economic, and sustainable development implications. Traditional sludge resource recovery typically includes energy recovery (biogas, heat, etc.) and material recovery (phosphorus, biochar, etc.), but traditional methods often suffer from low recovery efficiency, low economic benefits of recovered materials, and greenhouse gas emissions.

[0003] Research has discovered an extracellular polymer—polysaccharide sulfate—in activated sludge. Industrial-grade polysaccharide sulfate is widely used in industries such as printing and dyeing, papermaking, textiles, and rubber processing. Food / cosmetic-grade polysaccharide sulfate is mainly used as a thickener and antioxidant in food production and processing, as well as in cosmetics manufacturing. Pharmaceutical-grade polysaccharide sulfate can be used as a raw material for anticoagulant and antiviral drugs. Heparin, in particular, has highly effective anti-inflammatory, anticoagulant, and antiviral properties, and even exhibits the effect of inhibiting tumor cell metastasis. Commercially available heparin is a mucopolysaccharide extracted from porcine gastric mucosa, but due to its limited raw material and high market demand, its price remains high. Therefore, in the process of sludge resource recovery, it is of great significance to focus on the recovery of high-value substances and to find substances with similar biological activities to heparin. Summary of the Invention

[0004] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of this invention is to provide a method for the resource utilization of activated sludge; a second objective is to provide a heparinized polysaccharide obtained by this method; and a third objective is to provide the application of the sludge residue generated during this method in methanogenesis.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of this invention provides a method for the resource utilization of activated sludge, comprising the following steps:

[0007] 1) Wash the activated sludge to obtain precipitated sludge; mix the precipitated sludge with water to obtain a suspension; digest the suspension and separate it to obtain a first supernatant and sludge residue.

[0008] 2) The first supernatant was centrifuged sequentially with calcium salt solution and alcohol solvent to remove impurities, resulting in the first precipitate;

[0009] 3) Dissolve the first precipitate in an alkali metal acetate solution, add buffer solution, stir, and separate to obtain the second supernatant;

[0010] 4) The second supernatant is centrifuged with an alcohol solvent to remove impurities, yielding a second precipitate;

[0011] 5) Dissolve the second precipitate in water, add a polymer separating agent, let stand, and separate to obtain the third precipitate;

[0012] 6) Dissolve the third precipitate in an inorganic salt solution, shake, and separate to obtain the third supernatant;

[0013] 7) The third supernatant is centrifuged with an alcohol solvent to remove impurities, yielding a fourth precipitate;

[0014] 8) The fourth precipitate was purified to obtain heparinized polysaccharide.

[0015] Preferably, in step 1), the specific operation of cleaning the activated sludge is as follows: calcium chloride solution, phosphate buffer solution, and ultrapure water are added to the activated sludge in sequence, and centrifuged separately to obtain sludge precipitate. Adding different solutions can change the environmental conditions of the sludge, causing impurities (ions, inorganic salts, and suspended solids, etc.) in the sludge to dissolve or precipitate in different solutions, thereby being removed.

[0016] Preferably, in step 1), the volume ratio of calcium chloride solution, phosphate buffer solution and ultrapure water is 1:(1.8-2.2):(0.8-1.2); more preferably, the volume ratio of calcium chloride solution, phosphate buffer solution and ultrapure water is 1:(1.9-2.1):(0.9-1.1).

[0017] Preferably, in step 1), the concentration of the calcium chloride solution is 0.8–1.5 mol / L; more preferably, the concentration of the calcium chloride solution is 0.8–1.2 mol / L.

[0018] Preferably, in step 1), the concentration of the phosphate buffer solution (PBS) is 0.01–0.03 mol / L; more preferably, the concentration of the phosphate buffer solution is 0.01–0.02 mol / L.

[0019] Preferably, in step 1), the water content of the suspension is 92-99%.

[0020] Preferably, in step 1), the digestion temperature is 62–72°C; more preferably, the digestion temperature is 65–70°C.

[0021] Preferably, in step 1), the digestion time is 3 to 5 hours; more preferably, the digestion time is 3.5 to 4.5 hours.

[0022] After the activated sludge is washed, the sludge sediment is mixed with water and homogenized thoroughly to obtain a suspension (water content of 92-99%). The suspension is then heated and digested, which can change the structure and properties of the activated sludge, causing extracellular polymers (such as polysaccharides) to be released from the sludge into the solution. After centrifugation, the supernatant containing extracellular polymers, i.e., the first supernatant, can be obtained.

[0023] Preferably, in step 2), the specific operation for removing impurities is as follows: add calcium salt solution to the first supernatant, centrifuge to obtain supernatant I; add alcohol solvent to supernatant I until the alcohol volume concentration is 10-30%, stir, centrifuge to obtain supernatant II; add alcohol solvent to supernatant II until the alcohol volume concentration is 75-85%, let stand, and centrifuge to obtain the first precipitate.

[0024] Preferably, in step 2), the concentration of the calcium salt solution is 0.8–1.5 mol / L; more preferably, the concentration of the calcium salt solution is 0.8–1.2 mol / L.

[0025] Preferably, in step 2), the volume ratio of the calcium salt solution to the first supernatant is 1:(1.8-2.2); more preferably, the volume ratio of the calcium salt solution to the first supernatant is 1:(1.9-2.1).

[0026] Preferably, in step 2), stirring is carried out in a water bath at 0–4°C for 20–40 min; more preferably, stirring is carried out in a water bath at 0–4°C for 25–35 min.

[0027] Preferably, in step 2), the settling temperature is -15 to -25°C and the settling time is 8 to 15 hours; more preferably, the settling temperature is -17 to -23°C and the settling time is 10 to 12 hours.

[0028] Preferably, in step 2), the calcium salt solution includes a calcium chloride solution; and the alcohol solvent includes anhydrous ethanol.

[0029] The principle of impurity removal in the first supernatant is explained as follows:

[0030] (1) Alginate polysaccharide is a negatively charged polysaccharide molecule. The carboxyl group (COO-) in it carries a negative charge. When calcium chloride solution is added to the first supernatant, calcium ions will combine with the negative charge in the alginate polysaccharide to form a calcium salt precipitate. The formation of this precipitate will cause the alginate polysaccharide to precipitate out of the solution, which can be removed by centrifugation.

[0031] (2) Add ethanol to the supernatant until the volume concentration of ethanol is 20-30%. Within this concentration range, ethanol has the best solvent properties and can effectively desolubleate and precipitate nucleic acids. Stirring in the ice-water mixture can increase the efficiency of nucleic acid binding with ethanol, which helps to accelerate the formation and separation of the precipitate. The nucleic acid can be removed by centrifugation. This step can be repeated 2-3 times.

[0032] (3) Add ethanol to the supernatant until the volume concentration of ethanol is 75-85%. In a high concentration of ethanol solution, extracellular polymers such as polysaccharides and proteins are prone to precipitation. Stirring can further promote the reaction rate of extracellular polymers and ethanol. Low temperature environment is conducive to precipitation. By centrifugation, the extracellular polymer precipitate, i.e. the first precipitate, can be obtained.

[0033] Preferably, in step 3), the solid-liquid ratio of the first precipitate to the alkali metal acetate solution is 1 g:(8-12) mL; more preferably, the solid-liquid ratio of the first precipitate to the alkali metal acetate solution is 1 g:(9-11) mL. The amount of alkali metal acetate solution added must be sufficient to completely dissolve the precipitate.

[0034] Preferably, in step 3), the concentration of the alkali metal acetate solution is 8–12 wt%.

[0035] Preferably, in step 3), the alkali metal acetate solution includes at least one of sodium acetate and potassium acetate solution.

[0036] Preferably, in step 3), the volume ratio of the alkali metal acetate solution to the buffer solution is 1:(0.8-1.2); more preferably, the volume ratio of the alkali metal acetate solution to the buffer solution is 1:1.

[0037] Preferably, in step 3), the concentration of the buffer solution is 0.01–0.03 mol / L; more preferably, the concentration of the buffer solution is 0.01–0.02 mol / L.

[0038] Preferably, in step 3), the buffer solution includes a phenol-sodium acetate buffer solution.

[0039] Preferably, in step 3), stirring is carried out in a water bath at 0-4°C for 20-40 minutes; more preferably, stirring is carried out in a water bath at 0-4°C for 25-35 minutes.

[0040] Preferably, in step 4), the specific operation for removing impurities is as follows: Add alcohol solvent to the second supernatant until the alcohol volume concentration is 20-30%, let it stand, and centrifuge to obtain supernatant IV; add alcohol solvent to supernatant IV until the alcohol volume concentration is 75-85%, and centrifuge to obtain the second precipitate. This step can remove proteins.

[0041] Preferably, in step 4), the settling temperature is 2-6°C and the settling time is 10-20 min; more preferably, the settling temperature is 3-5°C and the settling time is 13-17 min.

[0042] Preferably, in step 5), the solid-liquid ratio of the second precipitate to water is 1 g:(8-12) mL; more preferably, the solid-liquid ratio of the second precipitate to water is 1 g:(9-11) mL. The amount of water added must be sufficient to completely dissolve the precipitate.

[0043] Preferably, in step 5), the concentration of the polymer separating agent is 3-6 wt%; more preferably, the concentration of the polymer separating agent is 3-5 wt%.

[0044] Preferably, in step 5), the volume ratio of the polymer separating agent to water is 1:(8-11); more preferably, the volume ratio of the polymer separating agent to water is 1:(8-10).

[0045] Preferably, in step 5), the polymer separating agent includes a hexadecanepyridine chloride solution.

[0046] Preferably, in step 5), the settling temperature is 20-25°C and the settling time is 8-15 hours; more preferably, the settling temperature is 20-25°C and the settling time is 10-12 hours.

[0047] Preferably, in step 6), the solid-liquid ratio of the third precipitate to the inorganic salt solution is 1 g:(8-12) mL; more preferably, the solid-liquid ratio of the third precipitate to the inorganic salt solution is 1 g:(9-11) mL.

[0048] Preferably, in step 6), the inorganic salt solution includes at least one of sodium chloride and potassium chloride solutions.

[0049] Preferably, in step 6), the concentration of the inorganic salt solution is 1.5–2.5 mol / L; more preferably, the concentration of the inorganic salt solution is 1.8–2.2 mol / L.

[0050] Preferably, in step 6), the oscillation temperature is 35-45°C and the time is 3-5 hours; more preferably, the oscillation temperature is 37-42°C and the time is 3.5-4.5 hours.

[0051] Preferably, in step 7), the specific operation for removing impurities is as follows: add alcohol solvent to the third supernatant until the alcohol concentration is 30-40%, let it stand, and centrifuge to obtain the fourth precipitate.

[0052] Preferably, in step 7), the settling temperature is 2-6°C and the settling time is 8-15 hours; more preferably, the settling temperature is 3-5°C and the settling time is 10-12 hours.

[0053] Preferably, in step 8), the purification process involves adding ethanol to the pulverized product until the ethanol concentration in the system is 55-65%, centrifuging to remove the precipitate, and then freeze-drying the supernatant to obtain the heparinized polysaccharide.

[0054] The second aspect of the present invention provides heparinized polysaccharides obtained by the resource utilization treatment method of activated sludge described in the first aspect of the present invention.

[0055] The third aspect of the present invention provides the application of sludge residue generated from the activated sludge resource utilization method described in the first aspect of the present invention in methanogenesis.

[0056] Compared with the prior art, the beneficial effects of the present invention are:

[0057] 1) The activated sludge resource utilization method provided by the present invention effectively removes impurities such as proteins and nucleic acids, and the polysaccharide product has low impurity content and high polysaccharide yield; the sludge residue generated in the process can also be used for fermentation to produce methane, realizing the recycling of sludge resources, which has important environmental, economic and sustainable development significance.

[0058] 2) The heparinized polysaccharide obtained by the resource-based treatment method of activated sludge provided by the present invention has similar hemocoagulability and anticancer potential to commercially available heparin, and has high application value. Attached Figure Description

[0059] Figure 1 This is a schematic diagram of the activated sludge resource utilization process in the embodiment;

[0060] Figure 2 The infrared spectra of heparinized polysaccharide (HP) and commercially available heparin (CH) are shown in the examples.

[0061] Figure 3 The results of the heparinized polysaccharide anti-cancer cell proliferation detection obtained in the examples;

[0062] Figure 4 The results of the detection of the anti-cancer cell migration effect of heparinized polysaccharides obtained in the examples are shown. Detailed Implementation

[0063] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0064] Example

[0065] The activated sludge used in this embodiment was taken from the return sludge of the secondary sedimentation tank of the Macau Peninsula Wastewater Treatment Plant. The concentration of suspended solids (MLSS) in the sludge was 33.4 g / L, the concentration of volatile suspended solids (MLVSS) was 20.6 g / L, and the pH value was 6.5. Figure 1 This is a schematic diagram of the activated sludge resource utilization process in the embodiment. Figure 1 The following steps were performed to extract heparinized polysaccharides from the activated sludge:

[0066] 1) After centrifuging the activated sludge at 10,000 rpm for 5 min, add 1 mol / L CaCl2 solution to make the sludge water content reach 99%. Then add 0.01 mol / L PBS and ultrapure water at one time to wash away the impurities in the activated sludge. Centrifuge the sludge separately to obtain precipitated sludge. The volume ratio of CaCl2 solution, PBS and ultrapure water is 1:2:1.

[0067] 2) Add ultrapure water to the precipitated sludge obtained in step 1) and stir it with a homogenizer to make the water content of the sludge suspension not less than 99%. The suspension is digested at 67°C for 4 hours. This process transfers the extracellular polymers in the activated sludge to the solution. Centrifuge to obtain the supernatant and sludge residue. The sludge residue can be used for fermentation to produce methane.

[0068] 3) Add 1 / 2 volume of 1mol / L CaCl2 solution to the supernatant obtained in step 2) to remove the alginate polysaccharide in the solution. Centrifuge to obtain the supernatant. The total sugar content in the separated precipitate is 85% by phenol-sulfuric acid method.

[0069] 4) Add anhydrous ethanol to the supernatant obtained in step 3) (so that the volume concentration of ethanol in the supernatant reaches 25%), stir in an ice-water bath at 4°C for 30 min, centrifuge to remove nucleic acid, retain the supernatant, and detect the nucleic acid content in the separated precipitate by ultraviolet absorption method. The nucleic acid content is 76-85%. Repeat this step 2-3 times to reduce the nucleic acid content in the supernatant to below 1%.

[0070] 5) Add anhydrous ethanol to the supernatant obtained in step 4) (so that the volume concentration of ethanol in the supernatant reaches 80%), place at -20℃ overnight, and centrifuge to obtain extracellular polymer precipitate;

[0071] 6) Dissolve the extracellular polymer precipitate obtained in step 5) in 10% sodium acetate solution, with a solid-liquid ratio of 1g:10mL for the extracellular polymer precipitate to 10% sodium acetate solution. Add the same volume of phenol-sodium acetate buffer solution (3.57g of phenol dissolved in 1L of 10% sodium acetate solution), stir in an ice-water bath at 4℃ for 30min, and centrifuge to obtain the supernatant.

[0072] 7) Add anhydrous ethanol to the supernatant obtained in step 6) (so that the volume concentration of ethanol in the supernatant reaches 25%), place it at 4°C for 15 min, centrifuge to remove protein, retain the supernatant, and detect the protein content in the precipitate by the Folin-phenol reagent method. If it is 85%, repeat this step 2 to 3 times to reduce the protein content in the supernatant to below 1%.

[0073] 8) Add anhydrous ethanol to the supernatant obtained in step 7) (so that the volume concentration of ethanol in the supernatant reaches 80%) to obtain a precipitate of polysaccharide sulfate ester enriched.

[0074] 9) Dissolve the polysaccharide sulfate precipitate obtained in step 8) in ultrapure water. The solid-liquid ratio of the polysaccharide sulfate precipitate to ultrapure water is 1 g: 10 mL. Add hexadecanepyridine chloride solution (CPC) (final mass concentration is 0.5%), incubate overnight at room temperature, and centrifuge to obtain heparin-CPC polymer.

[0075] 10) Dissolve the heparin-CPC polymer obtained in step 9) in 2 mol / L NaCl solution. The solid-liquid ratio of the heparin-CPC polymer to the NaCl solution is 1 g: 10 mL. Place the solution in a shaker at 40 °C for 4 h to allow the heparin-CPC polymer to fully dissociate. Centrifuge to obtain the supernatant.

[0076] 11) Add anhydrous ethanol to the supernatant obtained in step 10) (so that the volume concentration of ethanol in the supernatant reaches 35.5%), incubate overnight at 4°C, centrifuge to obtain heparin-like polysaccharide precipitate, and desalt, filter, dry and pulverize the precipitate to obtain heparinized polysaccharide product.

[0077] 12) Dissolve the heparinized polysaccharide product obtained in step 11) in ultrapure water. The solid-liquid ratio of the heparinized polysaccharide product to ultrapure water is 1g:10mL. Add anhydrous ethanol (to make the volume concentration of ethanol in the system reach 60%). Centrifuge to remove the polysaccharide precipitate containing a larger molecular weight, and obtain a heparinized polysaccharide solution containing a smaller molecular weight. Freeze-dry to obtain heparinized polysaccharide crystals with a yield of 5.0±0.8mg / g MLVSS.

[0078] Detection methods and results:

[0079] (1) Infrared spectroscopy analysis was performed on the heparinized polysaccharide crystals obtained in the examples and commercially available heparin (purchased from Shanghai Yuanye Biotechnology Co., Ltd.). Figure 2 The infrared spectra of heparinized polysaccharide (HP) and commercially available heparin (CH) in the examples are shown below. Figure 2 It can be seen that the heparinized polysaccharide in the embodiments is very similar to the position and shape of the peak of commercially available heparin, with only the peak intensity being different, indicating that the two have the same functional groups and chemical structures.

[0080] (2) The total sugar content, protein content, and heavy metal content of the obtained heparinized polysaccharide crystals were detected by sulfuric acid-phenol method, Folin-phenol reagent method, and ion chromatography, respectively. The results showed that the total sugar content in the heparinized polysaccharide crystals was 54.4±5.4% by mass, the protein content was as low as 5.6±1.1% by mass, and heavy metals were not detected. This indicates that the heparinized polysaccharide extracted from activated sludge in this embodiment has a high yield, high total sugar content, and low impurity content. This shows that activated sludge is rich in polysaccharides and has high resource utilization value. Combined with the activated sludge resource treatment method provided by this invention, the release and extraction efficiency of polysaccharides can be increased, impurities can be effectively removed during the treatment process, the obtained polysaccharide crystals have low protein content, and no heavy metals were detected, indicating good impurity removal effect.

[0081] (3) The titer of the heparin-like polysaccharide obtained in the example was 59.7±28.9 units / mg when the heparin bioassay method (see Chinese Pharmacopoeia 2020 edition, Part IV) was used, indicating that it has a coagulation function similar to heparin.

[0082] (4) The anti-cancer cell proliferation and migration effects of the obtained heparinized polysaccharide were detected and compared with those of commercially available heparin. Figure 3 The results of the heparinized polysaccharide assay for anti-cancer cell proliferation obtained in the examples are as follows. Figure 4 The results of the detection of the anti-cancer cell migration effect of heparinized polysaccharides obtained in the examples were obtained by... Figure 3 and Figure 4 It was found that low concentrations (≤500ppm) of heparinized polysaccharide and commercially available heparin had no significant effect on the proliferation and migration of normal cells (H9C2), but had a significant inhibitory effect on the proliferation and migration of cancer cells (MDA-MB-231), proving that the obtained heparinized polysaccharide has similar anti-cancer potential to commercially available heparin.

Claims

1. A method for the resource-based treatment of activated sludge, characterized in that, Includes the following steps: 1) Add calcium chloride solution, phosphate buffer solution and ultrapure water to activated sludge in sequence, and centrifuge them separately to obtain precipitated sludge; The precipitated sludge is mixed with water to obtain a suspension, which is then digested at 62-72℃ for 3-5 hours to separate the first supernatant and sludge residue. 2) The first supernatant was centrifuged sequentially with calcium salt solution and alcohol solvent to remove impurities, yielding the first precipitate; 3) Dissolve the first precipitate in an alkali metal acetate solution, add buffer solution, stir, and separate to obtain the second supernatant; 4) The second supernatant is centrifuged with an alcohol solvent to remove impurities, yielding a second precipitate; 5) Dissolve the second precipitate in water at a solid-liquid ratio of 1g: (8~12)mL, add a 3~6wt% hexadecanepyridine chloride solution, wherein the volume ratio of the hexadecanepyridine chloride solution to water is 1: (8~11), let stand, and separate to obtain the third precipitate; 6) Dissolve the third precipitate in an inorganic salt solution with a concentration of 1.5~2.5mol / L at a solid-liquid ratio of 1g: (8~12)mL, shake, and separate to obtain the third supernatant; 7) Add alcohol solvent to the third supernatant until the alcohol volume concentration is 30-40%, let stand, and centrifuge to obtain the fourth precipitate; 8) The fourth precipitate was purified to obtain heparinized polysaccharide.

2. The method according to claim 1, characterized in that, In step 1), the concentration of the calcium chloride solution is 0.8~1.5 mol / L; the concentration of the phosphate buffer solution is 0.01~0.03 mol / L; and the volume ratio of the calcium chloride solution, phosphate buffer solution, and ultrapure water is 1: (1.8~2.2): (0.8~1.2). And / or, the water content of the suspension is >92%.

3. The method according to claim 1, characterized in that, In step 2), the specific operation for removing impurities is as follows: Add a calcium salt solution to the first supernatant, centrifuge to obtain supernatant I; add an alcohol solvent to supernatant I until the alcohol volume concentration is 10-30%, stir, centrifuge to obtain supernatant II; add an alcohol solvent to supernatant II until the alcohol volume concentration is 75-85%, let stand, and centrifuge to obtain the first precipitate; wherein... The concentration of the calcium salt solution is 0.8~1.5 mol / L; the volume ratio of the calcium salt solution to the first supernatant is 1:(1.8~2.2); the stirring is carried out in a water bath at 0~4℃ for 20~40 min; the settling temperature is -15~-25℃ for 8~15 h; the calcium salt solution includes calcium chloride solution; the alcohol solvent includes anhydrous ethanol.

4. The method according to claim 1, characterized in that, In step 3), the solid-liquid ratio of the first precipitate to the alkali metal acetate solution is 1 g: (8~12) mL; the concentration of the alkali metal acetate solution is 8~12 wt%; the volume ratio of the alkali metal acetate solution to the buffer solution is 1: (0.8~1.2); the concentration of the buffer solution is 0.01~0.03 mol / L; and the buffer solution includes a phenol-sodium acetate buffer solution. And / or, the stirring is carried out in a water bath at 0~4℃ for 20~40 min.

5. The method according to claim 1, characterized in that, In step 4), the specific operation for removing impurities is as follows: add alcohol solvent to the second supernatant until the alcohol volume concentration is 20-30%, let it stand at 2-6℃ for 10-20 minutes, and centrifuge to obtain supernatant IV; add alcohol solvent to the supernatant IV until the alcohol volume concentration is 75-85%, and centrifuge to obtain the second precipitate.

6. The method according to claim 1, characterized in that, In step 5), the temperature for standing is 20~25℃ and the time is 8~15h.

7. The method according to claim 1, characterized in that, In step 6), the inorganic salt solution includes at least one of sodium chloride and potassium chloride solution; And / or, the oscillation temperature is 35~45℃; the time is 3~5h.

8. The method according to claim 1, characterized in that, In step 7), the temperature for standing is 2~6℃ and the time is 8~15h.

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