A method for extracting and purifying sialic acid from a fermentation broth

Abstract

This invention discloses a method for extracting and purifying sialic acid from fermentation broth, relating to the field of sialic acid extraction technology. The method involves filtering *E. coli* fermentation broth through a ceramic membrane, followed by gentle hydrolysis under high pressure with dry ice and CO2. The hydrolysate is then pH-adjusted with sodium citrate buffer, and a composite powder of magnesium aluminum hydrotalcite and nano-silica is added. Protein and pigment adsorption and removal, as well as heterogeneous nucleation template dispersion, are simultaneously achieved in the same stirring step. The sodium citrate buffer pre-dispersion step effectively reduces electrostatic agglomeration caused by the opposite surface charges of the two powders. The purified solution is then vacuum-concentrated to a low to medium concentration, followed by induced crystallization in a low-temperature antisolvent. After drying, spherical sialic acid particles are obtained. This extraction and purification method achieves one-step coupling of impurity removal and spherical crystallization, eliminating the need for separate decolorization, seed crystal addition, and extrusion granulation processes. It produces high-purity, high-flowability, and anti-caking spherical sialic acid powder with low concentration energy consumption.

Description

Technical Field

[0001] This invention relates to the field of sialic acid extraction technology, and more specifically to a method for extracting and purifying sialic acid from fermentation broth. Background Technology

[0002] Microbial fermentation is currently the mainstream route for the industrial production of sialic acid. The fermentation broth is hydrolyzed to obtain an aqueous solution containing sialic acid monomers, along with coexisting microbial proteins and colored polyphenolic impurities. These impurities act as heterogeneous nucleation sites during crystallization, inducing the formation of irregular crystals such as needles and plates.

[0003] The aforementioned irregular crystalline powder has low bulk density, large angle of repose, and is prone to moisture absorption and caking during storage. When used in food and pharmaceutical processing, it causes severe dust generation and poor flowability, necessitating an extrusion granulation process before processing. However, the granulation process leads to degradation and loss of sialic acid. It is known in the industry that preparing spherical sialic acid particles can fundamentally optimize powder flowability and eliminate the granulation step; however, existing processes for preparing spherical sialic acid have significant shortcomings in their supporting infrastructure.

[0004] Existing spherical crystallization processes rely on ultra-high concentration liquids and interfacial tension for crystallization, resulting in extremely high energy consumption for concentration. Furthermore, the process lacks simultaneous deep impurity removal methods, leading to the mixing and encapsulation of protein and pigment impurities within the spherical particles, significantly reducing the purity of the final product. Simultaneously, the industry faces two dual constraints in its existing supporting processes: first, the strong acid hydrolysis route, which generates large amounts of saline waste acid, resulting in high environmental disposal costs. Moreover, the strong acid environment easily degrades sialic acid molecules and introduces new colored impurities, further exacerbating impurity interference during the crystallization stage; second, the single adsorption-based impurity removal process can only remove impurities from the liquid and lacks the ability to control crystal form, resulting in irregular powder formation after crystallization.

[0005] In summary, the present invention aims to provide a sialic acid purification method that can simultaneously complete impurity removal and spherical crystallization, achieving purification and crystal form regulation in one step, and using a mild acid-free hydrolysis system to prepare high-purity spherical sialic acid powder under low concentration and energy consumption conditions.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for extracting and purifying sialic acid from fermentation broth, thereby resolving the issues raised in the background section.

[0008] I. This invention addresses the technical shortcomings of existing technologies where the impurity removal process and the spherical crystallization process are separated and can only be operated in steps. It designs a composite powder of magnesium aluminum hydrotalcite and nano-silica as an integrated crystal form regulating adsorbent. The design concept is as follows: Magnesium aluminum hydrotalcite possesses a layered intercalation structure with positively charged interlayers. It can specifically capture negatively charged denatured proteins and fermentation pigments in the hydrolysate through electrostatic adsorption, eliminating the source of irregular crystal nuclei formation. Nano-silica, uniformly dispersed in the liquid phase, is rich in silanol groups on its surface, providing uniform and ordered attachment and precipitation sites for sialic acid molecules. This allows crystals to grow orderly on the template surface even at low to medium concentrations, without the need to concentrate the solution to an ultra-high viscosity state. When combined, impurity removal and nucleation template dispersion are completed simultaneously in the same stirring process, eliminating the need for separate decolorization and seed crystal addition steps.

[0009] It should be noted that because the magnesium aluminum hydrotalcite layers carry positive charges and the nano silica surface carries negative charges, when the two are directly mixed, they are prone to agglomeration due to electrostatic attraction. This causes the adsorption sites of the magnesium aluminum hydrotalcite to be covered by SiO2 particles, resulting in the loss of its impurity removal ability. At the same time, the SiO2 particles cannot be evenly dispersed due to agglomeration, thus losing their template function.

[0010] To address this issue, the present invention adds a sodium citrate buffer pre-dispersion step before adding the composite powder, which stabilizes the pH of the system at 5.0-5.5, effectively weakening the surface charge difference between the two powders, avoiding agglomeration at the source, and enabling them to independently and stably perform the dual functions of adsorption and depurification and heterogeneous nucleation in the same liquid phase environment.

[0011] Furthermore, this invention employs dry ice-CO2 high-pressure, gentle hydrolysis instead of traditional strong acid hydrolysis. Dry ice sublimates into CO2 in a sealed reactor, creating a high-pressure, weakly acidic aqueous environment to complete the gentle depolymerization of polysialic acid. No external acids such as hydrochloric acid or sulfuric acid are introduced throughout the process, resulting in no large amounts of waste salt or acid. This also avoids the degradation of sialic acid and the introduction of new colored impurities caused by strong acid environments. After hydrolysis, dissolved CO2 can be removed simply by stirring at atmospheric pressure, without affecting the pH environment of the subsequent crystallization system.

[0012] Through the design of the buffer pre-dispersion coupled compound powder described above, this invention achieves the simultaneous one-step completion of impurity removal and spherical crystallization, and prepares high-purity spherical sialic acid powder under low concentration energy consumption conditions. This fundamentally solves the technical problem in the prior art where impurity removal and morphology control are mutually separated and require step-by-step operation.

[0013] II. A method for extracting and purifying sialic acid from fermentation broth, comprising the following steps: Step 1: Filter the E. coli fermentation broth through a ceramic membrane, collect the clear filtrate and transfer it to a closed high-pressure reactor. Add dry ice, seal and heat to 108-112℃ for hydrolysis. After hydrolysis, release the pressure, stir to remove dissolved CO2, and filter to obtain crude hydrolysate. Step 2: Add sodium citrate buffer to the crude hydrolysate to adjust the pH of the system to 5.0-5.5 and construct a weak buffer phase environment; Step 3: Add the composite powder of magnesium aluminum hydrotalcite and nano silica to the buffer-modified hydrolysate, stir to allow the magnesium aluminum hydrotalcite to adsorb protein and pigment impurities in the hydrolysate, and at the same time allow the nano silica particles to disperse in the liquid phase as heterogeneous nucleation templates; after stirring, filter and collect the purified pretreatment solution. Step 4: Vacuum concentrate the purified pretreatment solution to a sialic acid concentration of 200-280 g / L to obtain a medium-low concentration sialic acid concentrate; Step 5: Add the sialic acid concentrate to a low-temperature mixed antisolvent and stir to crystallize; after crystallization, filter and collect the crystallized product, and dry to obtain spherical sialic acid particles.

[0014] Preferably, the mass ratio of magnesium aluminum hydrotalcite to nano silica in the composite powder is 7:3; the amount of the composite powder added accounts for 3-4% of the total mass of the hydrolysate.

[0015] Preferably, the ceramic membrane in step 1 has a pore size of 50-200 nm; the ratio of dry ice to fermentation filtrate is 1 g: 130-155 mL; the hydrolysis temperature is 108-112℃, the hydrolysis time is 2-2.5 h, and the equilibrium pressure inside the high-pressure reactor is 0.22-0.35 MPa.

[0016] Preferably, the sodium citrate buffer salt in step 2 is food-grade sodium citrate, the pH of the system is adjusted to 5.0-5.5, and the stirring time is 10-15 min.

[0017] Preferably, the stirring temperature in step 3 is 50-60℃ and the stirring time is 20-40 min.

[0018] Preferably, the composite powder after adsorption saturation in step 3 is recycled after being soaked and washed with dilute alkali, rinsed with deionized water, and dried.

[0019] Preferably, the vacuum concentration temperature in step 4 is 60-75°C.

[0020] Preferably, the mixed antisolvent in step 5 is a mixture of anhydrous ethanol and isopropanol in a volume ratio of 3:7; the antisolvent temperature is 10-15℃; the concentrated droplet acceleration rate is 5-20 mL / min; the antisolvent volume is 4-10 times the volume of the concentrated liquid; the stirring speed is 350-450 r / min; and the crystallization time is 8-10 h.

[0021] The method for extracting and purifying sialic acid from fermentation broth provided in this invention has the following beneficial effects: (1) This invention uses magnesium aluminum hydrotalcite and nano silica to form a bifunctional composite powder, which simultaneously completes impurity adsorption and nucleation template dispersion in the same stirring process. Magnesium aluminum hydrotalcite electrostatically adsorbs and removes protein and pigment impurities, eliminating the source of heterogeneous crystal nuclei from the outset; nano silica, as a heterogeneous nucleation template, significantly reduces the supersaturation required for crystallization, allowing sialic acid to induce spherical growth at low to medium concentrations. This integrated design eliminates the need for separate decolorization and seed crystal addition processes, simplifying the production line and reducing equipment investment and labor costs.

[0022] (2) By adding a sodium citrate buffer pre-dispersion process, this invention effectively solves the technical problem that magnesium aluminum hydrotalcite and nano silica are prone to electrostatic agglomeration when directly mixed due to their opposite surface charges. The buffer system stabilizes the pH at 5.0-5.5, significantly reducing the surface charge difference between the two powders, allowing them to independently and stably perform the dual functions of adsorption and depurification and heterogeneous nucleation in the same liquid environment.

[0023] (3) This invention uses dry ice CO2 high-pressure mild hydrolysis to replace the traditional strong acid hydrolysis process. It does not introduce external acids such as hydrochloric acid and sulfuric acid. After hydrolysis, there is no large amount of waste salt or waste acid produced, and the wastewater treatment cost is greatly reduced. At the same time, the CO2 aqueous solution is weakly acidic and the hydrolysis conditions are mild, avoiding the degradation of sialic acid monomers and the problem of colored impurities by-products under strong acid conditions, and the purity of the finished product is significantly improved.

[0024] (4) This invention relies on the heterogeneous nucleation template effect of nano-silica to enable crystallization to be completed at low to medium concentrations without the need to concentrate the liquid to an ultra-high viscosity state. The energy consumption of concentration is significantly reduced compared with the high concentration process, while avoiding the problem of adhesion and agglomeration during the droplet addition of high viscosity liquid. The spherical particles have good uniformity and high batch-to-batch stability.

[0025] (5) The spherical sialic acid particles obtained by this invention have a dense and smooth surface, high bulk density, excellent flowability, and significantly better anti-caking performance than conventional irregular crystals. The powder can be directly used for tablet and solid compound food raw material processing without extrusion granulation, avoiding the thermal degradation loss of sialic acid during the thermal processing stage.

[0026] (6) The composite powder of the present invention can be recycled and reused after being washed with dilute alkali. Industrially, the powder can be recycled and reused, effectively controlling production costs. Attached Figure Description

[0027] Figure 1 This is a scanning electron microscope (SEM) image of the spherical sialic acid particles prepared in Example 3. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] To address the aforementioned technical problems, this invention provides a method for extracting and purifying sialic acid from fermentation broth, thereby resolving the issues raised in the background section.

[0030] I. Experimental Materials and Equipment raw material: Fermentation broth: Company's internal E. coli fermentation broth, polysialic acid concentration 25-35 g / L; Dry ice: Industrial food-grade dry ice; Sodium citrate: Food-grade sodium citrate buffer salt; Magnesium aluminum hydrotalcite: Commercially available pharmaceutical grade, CAS 11097-59-9, D50=2 μm; Nano silica: food-grade fumed silica, particle size 10-30 nm; Anhydrous ethanol: pharmaceutical grade; Isopropanol: Pharmaceutical grade; Dilute sodium hydroxide: industrial grade, prepared as a 3% aqueous solution; Deionized water: Prepared routinely in the workshop.

[0031] equipment: High-pressure reactors (0.5-50 L pressure-resistant temperature-controlled reactors), ceramic membrane filter units, vacuum concentrators and evaporators, low-temperature crystallization reactors, constant flow drip pumps, high-speed agitators, vacuum drying ovens, plate and frame filter presses, and vibrating screens.

[0032] II. Examples and Comparative Examples Example 1 This embodiment provides a method for extracting and purifying sialic acid from fermentation broth, including the following steps: (1) Pretreatment of fermentation broth: 100 L of Escherichia coli fermentation broth was filtered through a 100 nm ceramic membrane, and the clear filtrate was collected and transferred to a closed high-pressure reactor. Dry ice was added at a ratio of 1 g: 140 mL, the reactor was sealed, and the temperature was raised to 110 °C. Hydrolysis was carried out at this temperature for 2.2 h, and the equilibrium pressure inside the reactor was 0.28 MPa. After hydrolysis, the pressure was released, and the reactor was stirred at atmospheric pressure for 12 min to remove dissolved CO2. The solution was then filtered to obtain crude hydrolysate.

[0033] (2) Buffer pre-dispersion treatment: Add food-grade sodium citrate buffer salt to the crude hydrolysate, adjust the pH of the system to 5.2, stir at room temperature for 10 min to obtain buffer modified hydrolysate.

[0034] (3) One-step coupled simultaneous impurity removal and heterogeneous nucleation: The composite powder was prepared at a mass ratio of magnesium aluminum hydrotalcite to nano silica of 7:3, and the total amount of powder added was 3.5% of the total mass of the hydrolysate. The composite powder was added to the buffer-modified hydrolysate and stirred at 55°C for 30 min. After stirring, the mixture was filtered by plate and frame filter press, and the purified pretreatment liquid was collected.

[0035] (4) Medium and low concentration vacuum concentration: The purified pretreatment liquid is concentrated under vacuum at 65℃, and the sialic acid concentration is controlled at 240g / L to obtain medium and low concentration sialic acid concentrate.

[0036] (5) Low-temperature gradient directional spherical crystallization: The concentrated solution was added dropwise at a rate of 12 mL / min to a mixed antisolvent at 12℃. The antisolvent was a mixture of anhydrous ethanol and isopropanol in a volume ratio of 3:7, and the volume of the antisolvent was 5 times that of the concentrated solution. The mixture was stirred continuously at a stirring rate of 400 r / min for 9 h. After crystallization, the mixture was filtered, dried under vacuum at 55℃ for 14 h, and 40-110 μm particles were collected by sieving to obtain spherical sialic acid particles.

[0037] Example 2 The only difference between this embodiment and Example 1 is that in step (2), sodium citrate is used to adjust the pH of the system to 5.0; and in step (3), the amount of composite powder added is 3.0% of the total mass of the hydrolysate. The remaining steps and parameters are the same as in Example 1.

[0038] Example 3 The only difference between this embodiment and Example 1 is that in step (2), sodium citrate is used to adjust the pH of the system to 5.5; and in step (3), the amount of composite powder added is 4.0% of the total mass of the hydrolysate. The remaining steps and parameters are the same as in Example 1.

[0039] Comparative Example 1 The only difference between this comparative example and Example 1 is that step (2) of buffer pre-dispersion treatment is omitted, that is, sodium citrate buffer salt is not added to the crude hydrolysate, the pH is not adjusted, and the composite powder is added directly. The remaining steps and parameters are the same as in Example 1.

[0040] Comparative Example 2 The only difference between this comparative example and Example 1 is that in step (3), all the composite powder is replaced with magnesium aluminum hydrotalcite, that is, no nano silica is added. The amount of composite powder added is still 3.5% of the total mass of the hydrolysate, and the mass ratio of magnesium aluminum hydrotalcite to nano silica is 10:0. The remaining steps and parameters are the same as in Example 1.

[0041] Comparative Example 3 The only difference between this comparative example and Example 1 is that in step (3), all the composite powder is replaced with nano-silica, that is, magnesium aluminum hydrotalcite is not added. The amount of composite powder added is still 3.5% of the total mass of the hydrolysate, and the mass ratio of magnesium aluminum hydrotalcite to nano-silica is 0:10. The remaining steps and parameters are the same as in Example 1.

[0042] Comparative Example 4 The only difference between this comparative example and Example 1 is that in step (1), traditional strong acid hydrolysis is used instead of dry ice CO2 hydrolysis. Specifically, 0.2 mol / L sulfuric acid is added to the clarified filtrate after ceramic membrane filtration, the temperature is raised to 110℃ and kept at that temperature for 2.2 h for hydrolysis. After hydrolysis, the solution is neutralized to pH 7.0 with sodium hydroxide and filtered to obtain crude hydrolysate. Subsequent steps (2) to (5) are the same as in Example 1.

[0043] The key parameters of each embodiment and comparative example are shown in Table 1.

[0044] Table 1: Comparison of key parameters between the examples and comparative examples

[0045] III. Performance Testing and Results The products of Examples 1-3 and Comparative Examples 1-4 were tested, including: hydrolysis conversion rate (HPLC method), purity of sialic acid in the finished product, bulk density, angle of repose, number of days of accelerated caking at 37℃ / 75% relative humidity, particle morphology, residual silicon content, and overall yield of the entire process. The results are shown in Table 2.

[0046] Table 2: Performance Test Results of Examples and Comparative Examples

[0047] IV. Results Analysis The experimental data in Table 2 fully verify that the present invention systematically solves the technical problem of simultaneous impurity removal and spherical crystallization by using sodium citrate buffer pre-dispersion coupled with magnesium aluminum hydrotalcite-nano SiO2 composite powder.

[0048] (1) Regarding the key role of the buffer pre-dispersion process: The only difference between Comparative Example 1 and Example 1 is whether or not a buffer pre-dispersion treatment has been performed. The results show that in Comparative Example 1, because sodium citrate buffer salt was not added to adjust the pH, the magnesium aluminum hydrotalcite and nano SiO2 were directly mixed and electrostatic agglomeration occurred. The purity of the finished product was only 89.7%, and the particle morphology was irregular fragmented crystals with severe agglomeration.

[0049] In Examples 1-3, after pre-dispersion treatment with sodium citrate buffer, the pH of the system was stably controlled at 5.0-5.5, and both powders functioned stably and synchronously. The purity of the finished product reached 99.1%-99.5%, and the particle morphology was smooth and spherical. This fully demonstrates that the buffer pre-dispersion process is a key technical means to solve the problem of agglomeration of the two powders. Without this process, the dual function of the composite powder will be lost due to agglomeration.

[0050] (2) Regarding the irreplaceability of magnesium aluminum hydrotalcite: Comparative Example 2, which only added magnesium aluminum hydrotalcite without adding nano-SiO2, achieved a purity of 96.6%, but the particle morphology was irregular fragmented crystals, with a bulk density of only 0.41 g / mL, an angle of repose of 44°, and poor powder processing performance. This indicates that magnesium aluminum hydrotalcite only has the function of removing impurities and does not have the ability to control crystal morphology; it cannot be used alone to prepare spherical particles.

[0051] (3) Regarding the irreplaceability of nano-SiO2: Comparative Example 3, which only added nano-SiO2 without adding magnesium aluminum hydrotalcite, achieved a spherical morphology, bulk density, and angle of repose similar to the example, but the purity of the finished product was only 92.5%, and the particles contained a large number of colored impurities. This indicates that nano-SiO2 only has a template function but does not have the ability to remove impurities, and cannot obtain a high-purity product when used alone.

[0052] (4) Regarding the advantages of mild hydrolysis of dry ice with CO2: Comparative Example 4 used traditional sulfuric acid hydrolysis. Although the hydrolysis conversion rate was the same as that of the Example, the purity of the finished product was only 95.1%, the morphology was flaky crystals, and the total yield was 75.8%, which was significantly lower than that of the Example. This is because the strong acid environment caused the degradation of sialic acid and the addition of colored impurities, which aggravated the impurity interference in the crystallization stage.

[0053] (5) Regarding comprehensive performance and industrial value: The overall yield of the entire process in Examples 1-3 reached 85.1%-86.7%, which is significantly better than that of the comparative examples. The resulting spherical particles have a bulk density ≥0.56 g / mL, an angle of repose ≤34°, and accelerated caking ≥43 days. They have excellent powder processing performance and can be directly used for the processing of tablets and solid compound food raw materials without the need for extrusion granulation.

[0054] Conclusion: The method for extracting and purifying sialic acid from fermentation broth provided in this invention effectively weakens the electrostatic attraction between magnesium aluminum hydrotalcite and nano-SiO2 through a sodium citrate buffer pre-dispersion process, thus preventing agglomeration at its source. This allows both materials to simultaneously and stably perform their adsorption, depurification, and heterogeneous nucleation template functions in the same stirring process. Combined with gentle hydrolysis using dry ice and CO2, high-purity (≥99.0%), regularly spherical sialic acid particles with excellent powder processing properties are prepared under low-energy-consumption concentration conditions, demonstrating significantly superior overall performance compared to existing technologies.

[0055] 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 and purifying sialic acid from fermentation broth, characterized in that, Includes the following steps: Step 1: Filter the E. coli fermentation broth through a ceramic membrane, collect the clear filtrate and transfer it to a closed high-pressure reactor. Add dry ice, seal and heat to 108-112℃ for hydrolysis. After hydrolysis, release the pressure, stir to remove dissolved CO2, and filter to obtain crude hydrolysate. Step 2: Add sodium citrate buffer to the crude hydrolysate to adjust the pH of the system to 5.0-5.5 and construct a weak buffer phase environment; Step 3: Add the composite powder of magnesium aluminum hydrotalcite and nano silica to the buffer-modified hydrolysate, and stir to allow the magnesium aluminum hydrotalcite to adsorb protein and pigment impurities in the hydrolysate; at the same time, allow the nano silica particles to disperse in the liquid phase as heterogeneous nucleation templates. After stirring, filter and collect the purified pretreatment liquid; Step 4: Vacuum concentrate the purified pretreatment solution to a sialic acid concentration of 200-280 g / L to obtain a medium-low concentration sialic acid concentrate; Step 5: Add the sialic acid concentrate dropwise into a low-temperature mixed antisolvent and stir to crystallize; After crystallization, the crystallized product is collected by filtration and dried to obtain spherical sialic acid particles.

2. The method according to claim 1, characterized in that, The mass ratio of magnesium aluminum hydrotalcite to nano silica in the composite powder is 7:3; the amount of the composite powder added accounts for 3-4% of the total mass of the hydrolysate.

3. The method according to claim 1, characterized in that, The ceramic membrane in step 1 has a pore size of 50-200 nm; the ratio of dry ice to fermentation filtrate is 1 g: 130-155 mL; the hydrolysis temperature is 108-112℃, the hydrolysis time is 2-2.5 h; and the equilibrium pressure inside the high-pressure reactor is 0.22-0.35 MPa.

4. The method according to claim 1, characterized in that, The sodium citrate buffer salt mentioned in step 2 is food-grade sodium citrate. The pH of the system is adjusted to 5.0-5.5, and the stirring time is 10-15 min.

5. The method according to claim 1, characterized in that, The stirring temperature in step 3 is 50-60℃, and the stirring time is 20-40 min.

6. The method according to claim 1, characterized in that, After the composite powder is saturated by adsorption in step 3, it is washed by soaking in dilute alkali, rinsed with deionized water, dried, and then recycled for reuse.

7. The method according to claim 1, characterized in that, The vacuum concentration temperature in step 4 is 60-75℃.

8. The method according to claim 1, characterized in that, The mixed antisolvent in step 5 is a mixture of anhydrous ethanol and isopropanol in a volume ratio of 3:7; the temperature of the antisolvent is 10-15℃; the droplet acceleration rate of the concentrated liquid is 5-20mL / min; the volume of the antisolvent is 4-10 times the volume of the concentrated liquid; the stirring speed is 350-450 r / min; and the crystallization time is 8-10 h.

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