Purification method for efficiently producing fucose by using genetically engineered bacteria

Abstract

The invention provides a purification method for efficiently producing fucose by using genetically engineered bacteria, the preservation number of the genetically engineered bacteria is CCTCC NO: M 20252837, fermentation liquor of the genetically engineered bacteria is purified, and the purification method comprises the following specific steps: S1, taking the fermentation liquor, and filtering by using a ceramic membrane to obtain filtrate I; s2, adding a decolorizing agent into the filtrate I, stirring and decolorizing, and filtering through a filtering membrane to obtain filtrate II; s3, performing ultrafiltration on the filtrate II, performing top washing with pure water, and collecting all filtrate to obtain filtrate III; s4, sequentially carrying out cationic resin and anion resin combined chromatography on the filtrate III to obtain a chromatographic solution; s5, performing electrodialysis desalination on the chromatographic solution until the conductivity is 1t; the concentration is 100 [mu] s / cm; s6, concentrating the desalted solution, adding absolute ethyl alcohol, firstly heating and stirring, and then cooling until stirring, so as to obtain a turbid liquid; s7, the turbid liquid is filtered, solid is eluted with absolute ethyl alcohol, vacuum drying is conducted, and fucose is obtained. The purification method provided by the invention has the advantages of high purity, high yield and short period, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of fermentation product purification technology, specifically relating to a purification method for the efficient production of fucose using genetically engineered bacteria. Background Technology

[0002] Fucose has a wide range of applications: (1) It is used in the culture medium of biopharmaceutical antibody manufacturing companies, where fucose is used to regulate the content of fucose, the core of antibody N-glycosylation; (2) It is used in the raw materials of small molecule active pharmaceutical ingredients companies, where fucose is used as the core material of active pharmaceutical ingredients for subsequent synthesis; (3) It is used in food, cosmetics, and excipients.

[0003] However, the defects and shortcomings of existing technologies limit the application of L-fucose: (1) Existing traditional extraction and chemical production methods use non-renewable plant-derived resources, and the treatment with acids, alkalis, and organic reagents generates a large amount of environmentally unfriendly hazardous waste. (2) Traditional extraction and separation processes are complex and reduce product purity, resulting in high costs for high-quality products. Existing domestically produced L-fucose products are low-purity and yellowish, and their application scenarios and added value are far inferior to those of high-purity L-fucose.

[0004] Therefore, in addition to developing other high-yield methods for obtaining fucose, the purification process for the product is also extremely urgent. Summary of the Invention

[0005] In view of this, this invention utilizes the previously constructed accession number: CCTCC NO: M 20252837, accession date: December 10, 2025, depositary institution: China Center for Type Culture Collection (CCTCC), classification and naming: Escherichia coli The genetically engineered strain Fuc-WT22 provides an efficient purification technique to obtain high-purity, high-yield fucose while achieving high fucose fermentation yield.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A purification method for the efficient production of fucose using genetically engineered bacteria, wherein the genetically engineered bacteria has the preservation number CCTCC NO: M 20252837, and the purification object is the fermentation broth obtained by inoculating CCTCC NO: M 20252837 bacteria into a fermentation medium; the fermentation medium consists of: yeast extract powder 5 g / L, diammonium hydrogen phosphate 4 g / L, anhydrous potassium dihydrogen phosphate 13.5 g / L, magnesium sulfate heptahydrate 1.4 g / L, trace element solution 1 ml / L, calcium chloride 2 g / L, vitamin B 12.2 mg / L, citric acid 1 g / L, glucose 5 g / L, glycerol 10 g / L, ammonium sulfate 5 g / L, and PPE defoamer; The trace element solution consists of: NaCl 5 g / L, MnCl2·4H2O 4 g / L, ZnSO4·7H2O 1 g / L, sodium molybdate dihydrate 0.5 g / L, FeCl3·6H2O 4.75 g / L, boric acid 0.575 g / L, CuSO4·5H2O 0.4 g / L, and concentrated sulfuric acid 2.038 ml / L. The purification steps are as follows: S1. Filter the fermentation broth using a ceramic membrane to obtain filtrate I; S2. Add decolorizing agent to filtrate I, stir to decolorize, and then filter through a filter membrane to obtain filtrate II; S3. Ultrafiltration is performed on filtrate II, and the filtrate is washed with pure water to collect all the filtrate, thus obtaining filtrate III. S4. The filtrate III is subjected to cation exchange resin and anion exchange resin combined chromatography in sequence to obtain the chromatographic solution; S5. Perform electrodialysis to desalinate the chromatography solution so that the conductivity of the chromatography solution is <100μs / cm, to obtain the desalinated solution; S6. Concentrate the desalination solution until no obvious solvent remains, add anhydrous ethanol, heat and stir first, then cool and stir again to obtain a suspension. S7. Filter the suspension and wash the solid with anhydrous ethanol. Collect the solid and dry it under vacuum to obtain fucose.

[0007] Furthermore, the ceramic membrane described in step S1 has a pore size of 50~200nm, and the specific filtration method is as follows: First, the ceramic membrane was cleaned by circulating 1M NaOH solution; then it was washed with pure water until the pH was neutral; then the fermentation broth was poured in for filtration, and then it was washed with pure water; finally, all the filtrate was collected to obtain filtrate I.

[0008] Furthermore, the decolorizing agent mentioned in step S2 is activated carbon, and the mass-volume ratio of activated carbon to filtrate I is 1:(50~100). The pore size of the filter membrane is 0.4~0.5μm.

[0009] Furthermore, the ultrafiltration membrane used in step S3 has a molecular weight cutoff of 8~12KD and an ultrafiltration pressure of <0.2MPa.

[0010] In some specific embodiments, preferably, the cation exchange resin in step S4 is a 001×4 type cation exchange resin, and the anion exchange resin is a D301 type anion exchange resin.

[0011] Furthermore, the joint tomography is detailed below: First, activate the 001×4 type cation exchange resin with 1M HCl solution, then rinse with pure water until neutral. Next, activate the D301 type anion exchange resin with 1M NaOH solution, then rinse with pure water until neutral. Then, pour filtrate III into the 001×4 type cation exchange resin column, let it stand, elute with pure water first, then with 0.5M NaCl solution, and collect all the chromatographic solution. Then, pour the chromatographic solution into the D301 type anion exchange resin column, let it stand, elute with pure water first, then with 0.5M NaCl solution, and collect all the chromatographic solution. This completes the combined chromatography.

[0012] In some specific embodiments, preferably, the electrodialysis desalination in step S5 is as follows: First, clean the electrodialysis equipment until the conductivity is <10μs / cm. Then, add a 3% NaCl solution to the brine tank, add pure water to the filtrate tank, add chromatography solvent to the raw material tank, and finally start electrodialysis. The volume ratio of NaCl solution, pure water, and chromatography solvent is 1:1:1.

[0013] Furthermore, the volume ratio of anhydrous ethanol to concentrated liquid in step S6 is (3~5):1; Heating and stirring conditions: temperature 40~50℃, speed 150~250rpm, time 1.5~2.5h; Cooling and stirring conditions: temperature 0~4℃, speed 150~250rpm, time 5~7h.

[0014] Furthermore, the filtration described in step S7 is performed using a Buchner funnel; The vacuum drying conditions are: temperature 45~55℃, time 7~9h.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes a previously constructed genetically engineered bacterium (accession number CCTCC NO: M 20252837) to ferment and obtain fucose. Through exploration of purification processes, high-purity L-fucose was obtained (the product is white, with a purity of >98%, a yield of >65%, and a short purification cycle of only 5 days), which fully meets the requirements for large-scale industrial production. Attached Figure Description

[0016] Figure 1 The image shows the HPLC detection results of the fermentation broth in Example 1 of this invention.

[0017] Figure 2 This is a TLC detection result of the fermentation product in Example 1 of the present invention.

[0018] Figure 3 This is a graph showing the HPLC detection results of the purified product in Example 1 of the present invention. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0020] Example 1 This embodiment provides a purification method for obtaining fermentation broth using genetically engineered bacteria with accession number CCTCC NO: M 20252837, as detailed below: 1. Obtaining the fermentation broth 1.1 Culture medium preparation Seed culture medium composition: yeast extract 5g / L, soybean peptone 10g / L, NaCl 10g / L.

[0021] Fermentation medium composition: yeast extract powder FM885 5g / L, diammonium hydrogen phosphate 4g / L, anhydrous potassium dihydrogen phosphate 13.5g / L, magnesium sulfate heptahydrate 1.4g / L, trace element solution 1ml / L, calcium chloride 2g / L, vitamin B 12.2mg / L, citric acid 1g / L, glucose 5g / L, glycerol 10g / L, ammonium sulfate 5g / L, PPE defoamer.

[0022] The supplemental culture medium consists of: 250 g / L glucose, 20 g / L magnesium sulfate, 250 g / L glycerol, and 1 ml / L trace element solution.

[0023] The micronutrient solution in the fermentation culture and fed culture medium consisted of the following components: NaCl 5 g / L, MnCl2·4H2O 4 g / L, ZnSO4·7H2O 1 g / L, sodium molybdate dihydrate 0.5 g / L, FeCl3·6H2O 4.75 g / L, boric acid 0.575 g / L, CuSO4·5H2O 0.4 g / L, and concentrated sulfuric acid 2.038 ml / L.

[0024] 1.2 Seed liquid preparation First, sterilize the seed culture medium in a shake flask and cool it to room temperature. Then, inoculate the strain into the seed culture medium at an inoculation rate of 0.5% (w / v) in a clean bench and place it in a shaker for constant temperature shaking culture at 37°C. 600 Once the pH reaches 1.0, the primary seed culture medium is obtained. The seed culture medium is then sterilized in a shake flask and cooled to room temperature. In a clean bench, the primary seed culture medium is inoculated into the seed culture medium at an inoculation rate of 0.1% (v / v), and the flask is placed in a shaker and incubated at 37°C with constant temperature shaking until the OD reaches 1.0. 600 When the value reaches 1.0, the seed solution is obtained.

[0025] 1.3 Fermentation Process In a 5L fermenter, fermentation medium was added according to the fermentation medium formula and sterilized at 121℃ for 20 minutes. Seed culture was inoculated into the fermenter at a rate of 3% (v / v). The temperature was controlled at 37℃, pH 7.0, and dissolved oxygen ≥30%. Once the dissolved oxygen rapidly rose above 60%, the fermentation process was divided into four stages: logarithmic growth, protein expression, product synthesis, and substrate sugar consumption. By controlling the dissolved oxygen, rotation speed, feed rate, and temperature at different stages, high-yield fucose fermentation could be successfully achieved. The specific parameters for the four stages are as follows: Phase 1: Rotation speed 300 rpm, temperature 37℃, feeding rate 3.0 g / L, dissolved oxygen 50%, incubation time 8 h; Second stage: Rotation speed 550 rpm, temperature 25℃, feeding rate 4.5 g / L, dissolved oxygen 30%, incubation time 5 h; Third stage: Rotation speed 600 rpm, temperature 30℃, feeding rate 8.5 g / L, dissolved oxygen 30%, incubation time 32 h; Fourth stage: Rotation speed 600 rpm, temperature 30℃, feeding rate 2.0 g / L, dissolved oxygen 30%, incubation time 22 h.

[0026] During the second stage, lactose was added at a rate of 1 g / L to induce lactation for 5 hours.

[0027] OD was measured after the above fermentation stages were completed. 600 Value, when OD 600 When the temperature drops, centrifuge the mixture in the tank (7000 rpm, 30 min), collect the supernatant, and the fermentation is complete.

[0028] The fermentation results were tested (see results below). Figure 1 , 2 ), with a yield of 80g / L.

[0029] 2. Purification of fermentation broth 2.1 Ceramic membrane filtration Take 20L of the above fermentation supernatant (i.e., containing a total fucose content of 1600g) and filter it through a ceramic membrane to obtain filtrate I.

[0030] The ceramic membrane has a pore size of 100 nm, and the specific filtration method is as follows: The ceramic membrane equipment was first cleaned with 5L of 1M NaOH solution for 15 minutes; then it was cleaned with pure water until the pH was neutral (7±0.5); then the sample solution was poured in and ceramic membrane filtration was started. The filtrate was collected. After all the filtrate was filtered out (the internal volume of the equipment was <2L), pure water was added in 3 portions for top washing, 2L each time; all the filtrate was collected; after all the filtrate was collected, the raw solution end was tested and discarded if no product was found; the ceramic membrane equipment was cleaned with 1M NaOH and then stored.

[0031] 2.2 Decolorization Take the above filtrate I, add activated carbon at a mass-to-volume ratio of 1:100, stir at 300 rpm for 2 hours at room temperature, and then filter using a positive pressure filter with a 0.45 μm filter membrane to obtain filtrate II.

[0032] 2.3 Ultrafiltration To remove protein from the above filtrate II, use an ultrafiltration device: First, wash the ultrafiltration device with pure water until the pH value reaches 7. Pour filtrate II into the device and collect the filtrate. After all the filtrate has been filtered out (the volume inside the device is <2L), add pure water to wash the top three times, 2L each time, and collect all the filtrate to obtain filtrate III.

[0033] The ultrafiltration membrane has a throttling molecular weight of 10KD and an ultrafiltration pressure of <0.2MPa. After ultrafiltration, it is cleaned twice with 5L 0.5M NaOH and finally stored with 0.5M NaOH. 2.4 Combined cross-linking chromatography The above filtrate III was purified by combined chromatography using 001×4 cation exchange resin and D301 anion exchange resin: first, the 001×4 cation exchange resin was activated by rinsing with 100 L of 1M HCl solution, then rinsed with pure water until the pH of the receiving solution was neutral (pH 7.0±0.5), then the D301 anion exchange resin was activated by rinsing with 100 L of 1M NaOH solution, and then rinsed with pure water until the pH of the receiving solution was neutral (pH 7.0±0.5).

[0034] Then, slowly pour filtrate III into a 001X4 chromatography column. After pouring all of it in, let it stand for 10 minutes. Elute the product with pure water, collecting every 10 L of eluent. The collected eluent is then used to confirm the product by TLC. All fractions containing the product are combined. After confirming that the product has been completely eluted, elute with 0.5 M NaCl solution, collecting every 10 L of eluent, for a total of 100 L. Discard the eluent after confirming that there is no product by TLC. The first step of chromatography is now complete.

[0035] Finally, the first step chromatography solution was slowly poured into the D301 chromatography column, and the eluent was collected every 10 L. After all the solution was added, the product was eluted with pure water, and the eluent was collected every 10 L. The collected solution was confirmed by TLC to identify the product, and all components containing the product were combined. After confirming that the product was completely eluted, 0.5M NaCl was used for elution, and the eluent was collected every 10 L, for a total of 100 L. After TLC confirmed that there was no product, the solution was discarded. The combined ion-exchange chromatography was completed, and the chromatography solution was obtained.

[0036] 2.5 Electrodialysis desalination The above chromatography solution was desalted using an electrodialysis apparatus: First, the apparatus was rinsed with pure water until the conductivity of the feed tank and filtrate tank was <10 μS / cm. Then, 10 L of 3% NaCl solution was added to the brine tank, 10 L of pure water to the filtrate tank, and 10 L of the chromatography solution from the previous step to the feed tank. After all the solutions were added, electrodialysis desalination was started until the conductivity of the feed tank was <100 μS / cm. The liquid in the feed tank was then collected, and the filtrate tank was discarded after TLC analysis showed no product. Finally, 10 L of chromatography solution and 10 L of pure water were added to both the feed tank and filtrate tank, and the above operation was repeated until all the chromatography solution was desalted. All collected liquids were combined to obtain the desalted solution. The electrodialysis apparatus was cleaned with 3% NaCl and then stored in pure water.

[0037] 2.6 Concentration and Pulping Take the above desalination solution and concentrate it by rotary evaporation until no obvious solvent remains. Add 5L of anhydrous ethanol to the rotary evaporation flask (at this time, the volume ratio of anhydrous ethanol to concentrated solution is 5:1). After dissolving and clarifying, pour it into a 10L flask. Heat to 45℃ and stir for 2 hours (200 rpm). Then cool to 4℃ and stir for 6 hours (200 rpm) to obtain a suspension.

[0038] 2.7 Finished product drying The above suspension was filtered using a Buchner funnel, and the solid was washed with 1L of anhydrous ethanol. The solid was then collected and placed in a vacuum drying oven for drying (drying temperature 50℃, drying time 8h). The white solid after drying is fucose.

[0039] The final dried sample weighed 1049.6 g, with a purity of 99.1% and an overall yield of 65.6%. The entire processing cycle took 5 days. The purified product test results are shown below. Figure 3 .

[0040] Example 2 This embodiment provides a purification method, the purification object of which is the fermentation supernatant (processing volume 20L) in Example 1. The steps are basically the same as those in Example 1, except that: in step 2.1 the pore size of the ceramic membrane is 50nm, in step 2.2 the mass-volume ratio of neutral carbon to filtrate I is 1:50, and in step 2.6 the amount of anhydrous ethanol is 3L (at this time the volume ratio of anhydrous ethanol to concentrate is 3:1), and the rest remain unchanged.

[0041] The final dried sample weighed 1073.6g, with a purity of 99.6% and an overall yield of 67.1%. The entire processing cycle took 5 days.

[0042] Example 3 This embodiment provides a purification method, the purification object of which is the fermentation supernatant (processing volume 20L) in Example 1. The steps are basically the same as those in Example 1, except that: in step 2.1 the pore size of the ceramic membrane is 200nm, in step 2.2 the mass-volume ratio of neutral carbon to filtrate I is 1:200, and in step 2.6 the amount of anhydrous ethanol is 4L (at this time the volume ratio of anhydrous ethanol to concentrate is 4:1), and the rest remain unchanged.

[0043] The final dried sample weighed 1028.8g, with a purity of 99.0% and an overall yield of 64.3%. The entire processing cycle took 5 days.

[0044] Comparative Example 1 This comparative example provides a purification method for the fermentation supernatant (processing capacity 20L) from Example 1, as follows: 1.1 Centrifugation The fermentation supernatant was centrifuged using a tubular centrifuge (centrifuge speed 14000 r / min, pump speed 100) to obtain supernatant I.

[0045] 1.2 Membrane Filtration Take the above supernatant I and filter it using a positive pressure filter with a 0.45μm filter membrane to obtain filtrate I.

[0046] 1.3 Column Chromatography The above filtrate I was purified by chromatography using D301 resin: First, the D301 resin was activated by rinsing with 100 L of 1M NaOH solution, and then rinsed with pure water until the pH of the receiving solution was neutral (pH 7.0 ± 0.5). Then, filtrate I was slowly poured into the chromatography column, and after all the filtrate was poured in, it was allowed to stand for 10 min. The product was then eluted with pure water, and the eluent was collected every 10 L. The collected solution was used to confirm the product by TLC, and all fractions containing the product were combined. After confirming that the product was completely eluted, 0.5M NaCl was used for elution, and the eluent was collected every 10 L, for a total of 100 L. After confirming that there was no product by TLC, the eluent was discarded, and the chromatography was completed, yielding the chromatographic solution.

[0047] 1.4 Concentration and Pulping The steps are the same as in step 2.6 of Example 1.

[0048] 1.5 Finished product drying The steps are the same as 2.7 in Example 1.

[0049] Ultimately, this comparative study yielded 363.2g of dried sample, with a purity of 61.3% and an overall yield of 22.7%. The entire processing cycle took 8 days.

[0050] Comparative Example 2 This comparative example provides a purification method for the fermentation supernatant (processing capacity 20L) in Example 1. The specific steps are basically the same as those in Comparative Example 1, except that a nanofiltration desalination process is added after column chromatography in step 1.3. All other steps remain unchanged.

[0051] The nanofiltration desalination process is as follows: Take the chromatography solution and pour it into the nanofiltration machine for nanofiltration desalination (nanofiltration membrane molecular weight cutoff: 200 Da, nanofiltration pressure 1.0 MPa, nanofiltration endpoint judgment: concentrated liquid volume < 5 L, filtrate conductivity < 100 μs / cm).

[0052] Ultimately, this comparative example yielded 217.6g of dried sample, with a purity of 71.4% and an overall yield of 13.6%. The entire processing cycle took 10 days.

[0053] Comparative Example 3 This comparative example provides a purification method for the fermentation supernatant (processing capacity 20L) from Example 1, as follows: 1.1 Ceramic membrane filtration The steps are the same as in step 2.1 of Example 1.

[0054] 1.2 Column Chromatography The steps are the same as in Comparative Example 1, section 2.1.

[0055] 1.3 Nanofiltration Desalination The steps are the same as those in Comparative Example 2: the chromatography solution is poured into the nanofiltration machine for nanofiltration desalination (nanofiltration membrane molecular weight cutoff: 200 Da, nanofiltration pressure 1.0 MPa, nanofiltration endpoint judgment: concentrated liquid volume < 5 L, filtrate conductivity < 100 μs / cm).

[0056] 1.4 Concentration and Pulping The steps are the same as 2.6 in Example 1.

[0057] 1.5 Finished product drying The steps are the same as 2.7 in Example 1.

[0058] Ultimately, this comparative study yielded 385.6g of dried sample with a purity of 68.7% and an overall yield of 22.7%, with the entire processing cycle lasting 8 days.

[0059] Comparative Example 4 This comparative example provides a purification method for the fermentation supernatant (processing volume 20L) in Example 1. The specific steps are basically the same as those in Comparative Example 3, except that an ultrafiltration protein removal process is added after ceramic membrane filtration in step 1.1 (wherein, the ultrafiltration protein removal process is the same as step 2.3 in Example 1), and the rest remain unchanged.

[0060] Ultimately, this comparative study yielded 331.2 g of dried sample, with a purity of 61.2% and an overall yield of 20.7%. The entire processing cycle took 5 days.

[0061] Comparative Example 5 This comparative example provides a purification method for the fermentation supernatant (processing capacity 20L) in Example 1. The specific steps are basically the same as those in Comparative Example 4, except that the nanofiltration desalination process in the steps is replaced by the electrodialysis desalination process (wherein, the ultrafiltration protein removal process is the same as step 2.5 in Example 1), and the rest remain unchanged.

[0062] Ultimately, this comparative study yielded 1086.4g of dried sample with a purity of 91.8% and an overall yield of 67.9%, with the entire processing cycle taking 5 days.

[0063] Comparative Example 6 This comparative example provides a purification method for the fermentation supernatant (processing volume 20L) in Example 1. The specific steps are basically the same as in Example 1, except that in step 2.6, during concentration and pulping, anhydrous ethanol is replaced with the same volume of methanol, while the rest remain unchanged.

[0064] Ultimately, this comparative study yielded 180.8g of dried sample with a purity of 98.2% and an overall yield of 11.3%, with the entire processing cycle taking 5 days.

[0065] The purification processes and purification results of the above embodiments and comparative examples are detailed in Table 1.

[0066] Table 1. Details of purification processes and purification results for the examples and comparative examples.

[0067] As can be seen from Table 1, the purification process of the present invention has significant advantages in product purity, yield and purification time, which provides a good foundation for large-scale production.

[0068] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0069] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A purification method for the efficient production of fucose using genetically engineered bacteria, characterized in that, The genetically engineered bacteria has the preservation number CCTCC NO: M 20252837. The purification object is the fermentation broth obtained by inoculating CCTCC NO: M 20252837 bacteria into the fermentation medium. The purification steps are as follows: S1. Filter the fermentation broth using a ceramic membrane to obtain filtrate I; S2. Add decolorizing agent to filtrate I, stir to decolorize, and then filter through a filter membrane to obtain filtrate II; S3. Ultrafiltration is performed on filtrate II, and the filtrate is washed with pure water to collect all the filtrate, thus obtaining filtrate III. S4. The filtrate III is subjected to cation exchange resin and anion exchange resin combined chromatography in sequence to obtain the chromatographic solution; S5. Perform electrodialysis to desalinate the chromatography solution so that the conductivity of the chromatography solution is <100μs / cm, to obtain the desalinated solution; S6. Concentrate the desalination solution until no obvious solvent remains, add anhydrous ethanol, heat and stir first, then cool and stir again to obtain a suspension. S7. Filter the suspension and wash the solid with anhydrous ethanol. Collect the solid and dry it under vacuum to obtain fucose.

2. The purification method according to claim 1, characterized in that, The ceramic membrane described in step S1 has a pore size of 50~200nm, and the specific filtration method is as follows: First, the ceramic membrane was cleaned by circulating 1M NaOH solution; then it was washed with pure water until the pH was neutral; then the fermentation broth was poured in and filtered, and then washed with pure water; finally, all the filtrate was collected to obtain filtrate I.

3. The purification method according to claim 1, characterized in that, The decolorizing agent mentioned in step S2 is activated carbon, and the mass-volume ratio of activated carbon to filtrate I is 1:(50~100). The pore size of the filter membrane is 0.4~0.5μm.

4. The purification method according to claim 1, characterized in that, The ultrafiltration membrane used in step S3 has a molecular weight cutoff of 8~12KD and an ultrafiltration pressure of <0.2MPa.

5. The purification method according to claim 1, characterized in that, The cation exchange resin mentioned in step S4 is a 001×4 type cation exchange resin, and the anion exchange resin is a D301 type anion exchange resin.

6. The purification method according to claim 5, characterized in that, The joint tomography is as follows: First, activate the 001×4 type cation exchange resin with 1M HCl solution, then rinse with pure water until neutral. Next, activate the D301 type anion exchange resin with 1M NaOH solution, then rinse with pure water until neutral. Then, pour filtrate III into the 001×4 type cation exchange resin column, let it stand, elute with pure water first, then with 0.5M NaCl solution, and collect all the chromatographic solution. Then, pour the chromatographic solution into the D301 type anion exchange resin column, let it stand, elute with pure water first, then with 0.5M NaCl solution, and collect all the chromatographic solution. This completes the combined chromatography.

7. The purification method according to claim 1, characterized in that, The electrodialysis desalination in step S5 is as follows: First, clean the electrodialysis equipment until the conductivity is <10μs / cm. Then, add a 3% NaCl solution to the brine tank, add pure water to the filtrate tank, add chromatography solvent to the raw material tank, and finally start electrodialysis. The volume ratio of NaCl solution, pure water, and chromatography solvent is 1:1:

1.

8. The purification method according to claim 1, characterized in that, The volume ratio of anhydrous ethanol to concentrated liquid in step S6 is (3~5):1; Heating and stirring conditions: temperature 40~50℃, speed 150~250rpm, time 1.5~2.5h; Cooling and stirring conditions: temperature 0~4℃, speed 150~250rpm, time 5~7h.

9. The purification method according to claim 1, characterized in that, The filtration described in step S7 is performed using a Buchner funnel; The vacuum drying conditions are: temperature 45~55℃, time 7~9h.

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