High-purity and high-yield separation and purification method of N-acetylglucosamine

Abstract

The invention discloses a high-purity and high-yield separation and purification method of N-acetylglucosamine, and belongs to the technical field of biochemical engineering separation. The invention discloses a high-purity and high-yield separation and purification method of N-acetylglucosamine, which comprises the following steps: by taking fermentation liquor containing N-acetylglucosamine obtained by microbial fermentation as raw material liquor, performing flocculation pretreatment on the raw material liquor, and removing suspended particles, microorganisms, soluble macromolecular substances and the like through centrifugation or microfiltration, thereby obtaining the high-purity and high-yield separation and purification method of N-acetylglucosamine. And filtering to obtain clear filtrate. And performing concentration for three times, cooling and crystallizing the mixed solvent, and separating to obtain crude sugar. The raw sugar is re-dissolved and decolorized, and then is subjected to ion exchange desalination. And finally, performing concentration, cooling and crystallization, and performing forced air drying to obtain N-acetylglucosamine crystals with the purity of more than 99.5% and the total process yield of 75.0-87.2%.

Description

Technical Field

[0001] The invention relates to the field of biochemical separation technology, and more specifically to a method for separating and purifying N-acetylglucosamine with high purity and high yield. Background Art

[0002] N-acetylglucosamine (GlcNAc) is an important monosaccharide existing in nature. It has stable physical and chemical properties and is easily soluble in water, but difficult to dissolve in organic solvents such as ethanol. It is commonly found in the human body, various microorganisms, animals and plants, and plays an important role in human health. In addition, N-acetylglucosamine can be converted into glucosamine (GlcN) by direct deacetylation under weakly acidic conditions, but its stability is relatively low and it is often used in the form of complex salts. Glucosamine and N-acetylglucosamine have been used in the human body to detoxify the liver and kidneys, treat osteoarthritis, restore and maintain cartilage, reduce inflammation and protect the liver.

[0003] Traditional methods for producing glucosamine and N-acetylglucosamine include chitin hydrolysis and enzymatic bioconversion. As the market demand for glucosamine and N-acetylglucosamine continues to increase, traditional production methods have failed to effectively solve the problems of raw material supply, environmental protection, production efficiency and food safety, resulting in a gradual inability to meet people's needs. The raw materials for both methods are taken from shrimp and crab shells. On the one hand, it is difficult to make major breakthroughs in environmental protection and production efficiency. On the other hand, glucosamine and N-acetylglucosamine from shrimp and crab shells have the risk of causing allergic reactions in patients with seafood allergies in clinical applications. Therefore, food-safe glucosamine and N-acetylglucosamine from non-crustacean sources are products currently needed by consumers.

[0004] Microbial fermentation uses microorganisms as carriers to produce glucosamine and N-acetylglucosamine, which effectively overcomes the shortcomings of traditional production methods and has the advantages of low environmental pollution, low cost, high yield, mild reaction conditions, and short production cycle. In recent years, the direct conversion of glucose using recombinant Escherichia coli has reached a product concentration of 180g / L, but its endotoxin residues can cause food safety problems. However, recombinant Bacillus subtilis, Corynebacterium glutamicum and other microbial synthesis have also been developed and used in the production of this product.

[0005] Since the separation and purification of glucosamine and N-acetylglucosamine accounts for a high proportion of their production costs, the separation and purification patents of N-acetylglucosamine reported in the past still have problems such as low purification efficiency, serious environmental pollution, and no universal treatment process for raw materials from different sources. Based on this, it is necessary to develop corresponding universal, high-purity and high-yield separation and purification methods for different raw materials.

[0006] Therefore, providing a high-purity and high-yield separation and purification method for N-acetylglucosamine is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a method for separating and purifying N-acetylglucosamine with high purity and high yield. The method uses fermentation broth rich in N-acetylglucosamine as raw material liquid, can efficiently extract and obtain high-purity N-acetylglucosamine solid powder and / or high-purity crystals, and solves the defects existing in the prior art such as low yield, high energy consumption, high pollution, poor process versatility, etc. in the extraction process.

[0008] In order to achieve the above object, the present invention adopts the following technical solution: The purpose of the present invention is to provide a method for separating and purifying N-acetylglucosamine. The method uses fermentation liquid containing N-acetylglucosamine as a raw material, sequentially performs flocculation, microfiltration, concentration, solvent cooling crystallization, crystal redissolution and decolorization, desalination, concentration cooling crystallization and drying treatment on the raw material liquid, and obtains N-acetylglucosamine crystals with a purity of ≥99.5%, and the total yield of the route is 75.0-87.2%.

[0009] A method for separating and purifying N-acetylglucosamine with high purity and high yield comprises the following steps: (1) adding a flocculant to a raw material solution containing N-acetylglucosamine for flocculation; (2) performing solid-liquid separation on the solution after flocculation in step (1) to obtain a clarified N-acetylglucosamine sterilization solution and solid waste; (3) concentrating the clarified N-acetylglucosamine sterilized liquid obtained in step (2) and cooling it for crystallization, and performing solid-liquid separation after crystallization to obtain N-acetylglucosamine crude sugar and residual mother liquor; (4) using pure water to redissolve the N-acetylglucosamine crude sugar obtained in step (3) to obtain an N-acetylglucosamine aqueous solution with a concentration of 30-50%, and using an adsorbent to decolorize the N-acetylglucosamine aqueous solution at 20-50° C. and a pH of 4-10 for 0.5-5 h, and after the decolorization, performing solid-liquid separation to obtain an N-acetylglucosamine decolorized solution and an adsorbent; (5) desalting the N-acetylglucosamine decolorizing solution obtained in step (4) to obtain N-acetylglucosamine desalted solution; the desalting method is ion exchange chromatography; (6) Concentrating the N-acetylglucosamine desalted liquid obtained in step (5) and cooling it for crystallization, and performing solid-liquid separation after crystallization to obtain N-acetylglucosamine crystals and residual clear liquid; the residual clear liquid is refluxed to step (5); the N-acetylglucosamine crystals are washed with an ethanol aqueous solution to obtain high-purity N-acetylglucosamine crystals; (7) Drying the high-purity N-acetylglucosamine crystals obtained in step (6); the drying method is air drying.

[0010] The raw material liquid is the fermentation liquid obtained by microbial fermentation.

[0011] The flocculant in step (1) is a food-grade safe flocculant, including but not limited to one or a combination of two or more of aluminum sulfate, ferric chloride, ferrous sulfate, ferric sulfate, polyferric sulfate, polyaluminum chloride, gelatin, sodium alginate, polyacrylamide, lignin sulfonate, chitin and their derivatives.

[0012] The added amount of the flocculant is 0.1-0.6% of the dry weight of the biomass in the raw material liquid; when the flocculants are used in combination, the proportion of each component flocculant is not less than 10%.

[0013] The flocculation method has an operating temperature of 40-70° C. and a flocculation time of 1-10 hours.

[0014] In step (2), the solid waste obtained by solid-liquid separation is dried by air drying and used as feed protein to improve the overall economic benefits; the solid-liquid separation method includes but is not limited to microfiltration or centrifugation; the microfiltration component is tubular, plate-and-frame or roll-type; the microfiltration membrane material is an organic polymer membrane or a ceramic membrane; the microfiltration method is cross-flow filtration; and the centrifugation uses a disc centrifuge.

[0015] The concentration method in step (3) is vacuum concentration, flash concentration or multiple-effect evaporation concentration; the solid-liquid separation method is vacuum filtration or centrifugation; the residual mother liquor obtained by batch separation is recrystallized twice, and the solid-liquid separation operation is repeated to finally obtain three-crystallized crude sugar.

[0016] The concentration and crystallization method is (a) or (b): (a) the clarified N-acetylglucosamine sterilization solution is concentrated at 45-80°C to obtain an N-acetylglucosamine concentrated solution with a concentration of 30-50%, and crystallization is performed; (b) the clarified N-acetylglucosamine sterilization solution is concentrated at 45-80°C to obtain an N-acetylglucosamine concentrated solution with a concentration of 30-50%, a food safety solvent with a mass fraction of 5-50% is added, and crystallization is performed; the solvent includes but is not limited to anhydrous ethanol, anhydrous methanol, ethylene glycol, and isopropanol; the cooling rate of the crystallization is 1-10°C / h; the crystallization stirring rate is 0-500rpm; when the temperature is cooled to 20-40°C, 0.5-3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) is added; the crystallization temperature is maintained at 5-10°C; and the crystallization time is maintained at 5-20h.

[0017] The adsorbent obtained by solid-liquid separation in step (4) can be regenerated by 1-5% sodium hydroxide and 1-5% hydrochloric acid for reuse of the adsorbent, thereby saving purification costs; the adsorbent includes but is not limited to activated carbon, macroporous decolorizing resin, diatomaceous earth or bentonite; the amount of activated carbon added is 0.5-2% of the mass of the N-acetylglucosamine aqueous solution; the amount of macroporous decolorizing resin added is 5-20% of the mass of the N-acetylglucosamine aqueous solution; the amount of diatomaceous earth added is 2-10% of the mass of the N-acetylglucosamine aqueous solution; and the amount of bentonite added is 2-10% of the mass of the N-acetylglucosamine aqueous solution.

[0018] The ion exchange chromatography in step (5) uses anion exchange resin and cation exchange resin to perform bipolar resin chromatography desalting.

[0019] The types of ion exchange resins used in the ion exchange chromatography and electrodeionization include but are not limited to styrene resins (732 resin, 717 resin), acrylic resins (D150 resin, D151 resin), and the resin form is macroporous or gel-type; the ion exchange resin is used in the form of a filling column in the ion exchange chromatography.

[0020] The ion exchange chromatography is in the form of a cation-anion exchange column system or an anion-cation exchange column system; and the equipment is in the form of a fixed bed or a simulated moving bed or a continuous moving bed.

[0021] The final conductivity of the desalted liquid obtained by the desalination method is 0.1-0.5 mS / cm.

[0022] In step (6), the N-acetylglucosamine desalted liquid is concentrated to obtain an N-acetylglucosamine concentrated liquid with a concentration of 40-70%. The solid-liquid separation method is vacuum filtration, and the separated N-acetylglucosamine crystals are washed with an ethanol aqueous solution with a mass fraction of 60-90%. The concentration method is one or more of vacuum evaporation concentration, flash concentration, multi-effect evaporation concentration or reverse osmosis membrane concentration.

[0023] The N-acetylglucosamine desalted liquid is concentrated at 45-80°C; the crystallization cooling rate is 5-20°C / h; the crystallization stirring rate is 0-400rpm; when the temperature is lowered to 20-40°C, 0.5-3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) is added; the crystallization temperature is maintained at 3-10°C; and the crystallization time is maintained for 5-20h.

[0024] The air drying temperature in step (7) is 40-80°C.

[0025] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a high-purity and high-yield separation and purification method for N-acetylglucosamine, which can obtain high-yield, low-pollution, low-energy consumption, and high-purity N-acetylglucosamine products, and has process versatility for raw material liquids from different sources, and has great prospects for industrial scale-up production. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1 The recombinant Corynebacterium glutamicum ΔnagA / BΔ1dhΔcg12642Δzwf-pec-BeS-B-CeA strain provided by the patent publication CN114250188A was used for fermentation in a 5L fermenter to obtain an N-acetylglucosamine fermentation broth with a concentration of 120 g / L, which was used as the raw material liquid for the separation and purification step.

[0028] Take 0.1% of the dry weight of the biomass in the raw material solution (the dry weight is the weight of the dried solid obtained after centrifugation and filtration of the raw material solution) of ionic polyacrylamide, add it to the raw material solution containing N-acetylglucosamine at 40°C, dissolve it and flocculate it for 10 hours. Subsequently, the flocculated raw material solution is cross-flow microfiltered through a tubular ceramic membrane to obtain a clarified N-acetylglucosamine sterilized solution. Concentrate under reduced pressure at 50°C to concentrate the sterilized solution to 300g / L, and cool it to 20°C at a cooling rate of 1°C / h at a stirring speed of 100rpm. At this time, add 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine), and then keep it at 5°C for 5h and filter it under reduced pressure to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 is concentrated and recrystallized, and filtered to obtain crude sugar 2 and residual mother liquor 2; the residual mother liquor 2 is then concentrated and recrystallized, and filtered to obtain crude sugar 3 and residual mother liquor 3. Next, the crude N-acetylglucosamine obtained by three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 300 g / L. 0.5% activated carbon was added to the solution, and after decolorization for 0.5 h at 20°C and pH 4, it was filtered under reduced pressure to obtain the N-acetylglucosamine decolorized solution. The decolorized solution was chromatographed by a "732-717" ion exchange resin series system until the conductivity of the discharge liquid was less than 0.1 mS / cm. Then, the desalted solution was concentrated to 400 g / L under reduced pressure at 50°C, and cooled to 20°C at a cooling rate of 5°C / h at a stirring speed of 50 rpm. At this time, 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the solution was kept at 3°C ​​for 5 h and then filtered under reduced pressure. The crystals obtained by filtration were washed with a 60% ethanol aqueous solution and dried in a blast drying oven at 40° C. to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 78.5%.

[0029] The total yield is calculated as follows: the mass of N-acetylglucosamine with a purity greater than 99.5% finally obtained / the mass of N-acetylglucosamine in the raw material solution without any treatment.

[0030] Example 2 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.3% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after the raw material liquid is centrifuged and filtered) of aluminum sulfate was added to the raw material liquid containing N-acetylglucosamine at 50°C to dissolve and flocculate for 5h. Subsequently, the flocculated raw material liquid was cross-flow microfiltered through a tubular ceramic membrane to obtain a clarified N-acetylglucosamine sterilized liquid. The sterilized liquid was concentrated under reduced pressure at 50°C to 400g / L, and cooled to 30°C at a cooling rate of 5°C / h at a stirring speed of 300rpm. At this time, 1.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added, and then kept at 10°C for 10h and then filtered under reduced pressure to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and filtered to obtain crude sugar 2 and residual mother liquor 2; the residual mother liquor 2 was then concentrated and recrystallized, and filtered to obtain crude sugar 3 and residual mother liquor 3. Then, the N-acetylglucosamine crude sugar obtained by three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 400 g / L. 2% activated carbon was added to the solution, and after decolorization for 2.5 hours at 35°C and pH 7, it was filtered under reduced pressure to obtain N-acetylglucosamine decolorized liquid. The decolorized liquid was chromatographed by a "717-732" ion exchange resin series system until the conductivity of the discharge liquid was less than 0.5 mS / cm. Then, the desalted liquid was concentrated under reduced pressure at 50°C to 500 g / L, and cooled to 30°C at a cooling rate of 10°C / h at a stirring speed of 250 rpm. At this time, 1.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 10°C for 10 hours before being filtered under reduced pressure. The filtered crystals were washed with 75% ethanol aqueous solution and dried in a blast drying oven at 60°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 77.1%.

[0031] Example 3 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.6% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after centrifugation and filtration of the raw material liquid) of ferric chloride was added to the raw material liquid containing N-acetylglucosamine at 70°C to dissolve and flocculate for 1h. Subsequently, the flocculated raw material liquid was cross-flow microfiltered through a plate-and-frame ceramic membrane to obtain a clarified N-acetylglucosamine sterilized liquid. The sterilized liquid was concentrated to 500g / L by flash evaporation, and cooled to 40°C at a cooling rate of 10°C / h at a stirring speed of 500rpm. At this time, 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added, and then vacuum filtered after keeping at 5°C for 15h to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and filtered to obtain crude sugar 2 and residual mother liquor 2; the residual mother liquor 2 was then concentrated and recrystallized, and filtered to obtain crude sugar 3 and residual mother liquor 3. Then, the N-acetylglucosamine crude sugar obtained by three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 500 g / L. 5% of macroporous decolorizing resin was added to the solution, and after decolorization for 5 hours at 50°C and pH 10, it was filtered under reduced pressure to obtain N-acetylglucosamine decolorized liquid. The decolorized liquid was chromatographed by a simulated moving bed system of "732-D150" ion exchange resin until the conductivity of the discharged liquid was less than 0.1 mS / cm. Then, the desalted liquid was concentrated to 600 g / L by flash evaporation, and cooled to 40°C at a cooling rate of 15°C / h at a stirring speed of 500 rpm. At this time, 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 3°C ​​for 15 hours before being filtered under reduced pressure. The crystals obtained by filtration were washed with 90% ethanol aqueous solution and dried in a forced air drying oven at 80°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 80.1%.

[0032] Example 4 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.1% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after centrifugation and filtration of the raw material liquid) of ferrous sulfate was added to the raw material liquid containing N-acetylglucosamine at 40°C to dissolve and flocculate for 10 hours. Subsequently, the flocculated raw material liquid was sterilized by a plate-and-frame ceramic membrane to obtain a clarified N-acetylglucosamine sterilized liquid. The sterilized liquid was concentrated to 300 g / L by flash evaporation, and cooled to 20°C at a cooling rate of 1°C / h at a stirring speed of 100 rpm. At this time, 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added, and then the solution was kept at 10°C for 20 hours and then filtered under reduced pressure to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and the crude sugar 2 and the residual mother liquor 2 were obtained by filtration; the residual mother liquor 2 was then concentrated and recrystallized, and the crude sugar 3 and the residual mother liquor 3 were obtained by filtration. Then, the N-acetylglucosamine crude sugar obtained by the three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 300 g / L. 20% of macroporous decolorizing resin was added to the solution, and after decolorization for 5 hours at 20°C and pH 7, it was filtered under reduced pressure to obtain the N-acetylglucosamine decolorized solution. The decolorized solution was chromatographed by a simulated moving bed system until the conductivity of the discharge liquid was less than 0.5 mS / cm. Then, the desalted solution was concentrated to 400 g / L by flash evaporation, and cooled to 20°C at a cooling rate of 20°C / h at a stirring speed of 50 rpm. At this time, 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 10°C for 20 hours before being filtered under reduced pressure. The crystals obtained by filtration were washed with 90% ethanol aqueous solution and dried in a blast drying oven at 40°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 80.6%.

[0033] Example 5 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.3% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after the raw material liquid is centrifuged and filtered) of iron sulfate was added to the raw material liquid containing N-acetylglucosamine at 50°C to dissolve and flocculate for 5h. Subsequently, the flocculated raw material liquid was cross-flow microfiltered through a rolled organic microfiltration membrane to obtain a clarified N-acetylglucosamine sterilized liquid. The sterilized liquid was concentrated to 400g / L by single-effect evaporation, and cooled to 30°C at a cooling rate of 5°C / h at a stirring speed of 300rpm. At this time, 1.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added, and then kept at 5°C for 5h and then filtered under reduced pressure to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and filtered to obtain crude sugar 2 and residual mother liquor 2; the residual mother liquor 2 was then concentrated and recrystallized, and filtered to obtain crude sugar 3 and residual mother liquor 3. Then, the N-acetylglucosamine crude sugar obtained by three crystallizations was re-dissolved in pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 400 g / L. 2% diatomaceous earth was added to the solution, and after decolorization for 0.5 h at 35°C and pH 4, it was filtered under reduced pressure to obtain N-acetylglucosamine decolorized liquid. The decolorized liquid was chromatographed by a continuous moving bed system until the conductivity of the discharge liquid was less than 0.1 mS / cm. Then, the desalted liquid was concentrated to 500 g / L by single-effect evaporation, and cooled to 30°C at a cooling rate of 5°C / h at a stirring speed of 250 rpm. At this time, 1.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 3°C ​​for 5 hours before being filtered under reduced pressure. The filtered crystals were washed with 75% ethanol aqueous solution and dried in a blast drying oven at 60°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 77.0%.

[0034] Example 6 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.6% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after the raw material liquid is centrifuged and filtered) of polyferric sulfate was added to the raw material liquid containing N-acetylglucosamine at 70°C to dissolve and flocculate for 10 hours. Subsequently, the flocculated raw material liquid was cross-flow microfiltered through a rolled organic microfiltration membrane to obtain a clarified N-acetylglucosamine sterilized liquid. The sterilized liquid was concentrated to 500 g / L by single-effect evaporation, and cooled to 40°C at a cooling rate of 10°C / h at a stirring speed of 500 rpm. At this time, 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added, and then vacuum filtered after keeping at 10°C for 10 hours to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and filtered to obtain crude sugar 2 and residual mother liquor 2; the residual mother liquor 2 was then concentrated and recrystallized, and filtered to obtain crude sugar 3 and residual mother liquor 3. Then, the N-acetylglucosamine crude sugar obtained by three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 500 g / L. 10% diatomaceous earth was added to the solution, and after decolorization for 5 hours at 35°C and pH 10, it was filtered under reduced pressure to obtain N-acetylglucosamine decolorized liquid. The decolorized liquid was chromatographed by a continuous moving bed system until the conductivity of the discharge liquid was less than 0.5 mS / cm. Then, the desalted liquid was concentrated to 600 g / L by single-effect evaporation, and kept at 10°C for 10 hours at a stirring speed of 500 rpm. At this time, 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the solution was kept at 8°C for 18 hours and then filtered under reduced pressure. The crystals obtained by filtration were washed with 90% ethanol aqueous solution and dried in a forced air drying oven at 80° C. to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 75.0%.

[0035] Example 7 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.02% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after the raw material liquid is centrifuged and filtered) of ionic polyacrylamide and 0.18% of polyaluminum chloride were added to the raw material liquid containing N-acetylglucosamine at 40°C to dissolve and flocculate for 10 hours. Subsequently, the flocculated raw material liquid was centrifuged by a disc centrifuge to obtain a clarified N-acetylglucosamine sterilized liquid. The sterilized liquid was concentrated to 300 g / L by multi-effect evaporation and cooled to 20°C at a cooling rate of 1°C / h at a stirring speed of 100 rpm. At this time, 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added, and then kept at 5°C for 15 hours and then filtered under reduced pressure to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and filtered to obtain crude sugar 2 and residual mother liquor 2; the residual mother liquor 2 was then concentrated and recrystallized, and filtered to obtain crude sugar 3 and residual mother liquor 3. Then, the N-acetylglucosamine crude sugar obtained by three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 300 g / L. 2% bentonite was added to the solution, and after decolorization at 50°C and pH 7 for 0.5h, it was filtered under reduced pressure to obtain N-acetylglucosamine decolorized liquid. The decolorized liquid was chromatographed by a simulated moving bed system of "732-D151" ion exchange resin until the conductivity of the discharged liquid was less than 0.1mS / cm. Then, the desalted liquid was concentrated to 400g / L by multi-effect evaporation, and cooled to 20°C at a cooling rate of 15°C / h at a stirring speed of 50rpm. At this time, 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 3°C ​​for 15 hours before being filtered under reduced pressure. The filtered crystals were washed with a 60% ethanol aqueous solution and dried in a blast drying oven at 40°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 76.2%.

[0036] Example 8 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.1% gelatin and 0.1% polyaluminium chloride of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after the raw material liquid is centrifuged and filtered) are added to the raw material liquid containing N-acetylglucosamine at 50°C to dissolve and flocculate for 10 hours. Subsequently, the flocculated raw material liquid is centrifuged by a disc centrifuge to obtain a clarified N-acetylglucosamine sterilized liquid. The sterilized liquid is concentrated to 400g / L by multi-effect evaporation, and cooled to 30°C at a cooling rate of 5°C / h at a stirring speed of 300rpm. At this time, 1.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) is added, and then filtered under reduced pressure after being kept at 10°C for 20 hours, thereby obtaining N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and the crude sugar 2 and the residual mother liquor 2 were obtained by filtration; the residual mother liquor 2 was then concentrated and recrystallized, and the crude sugar 3 and the residual mother liquor 3 were obtained by filtration. Then, the N-acetylglucosamine crude sugar obtained by the three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 400 g / L. 10% bentonite was added to the solution, and after decolorization for 5 hours at 50°C and pH 4, it was filtered under reduced pressure to obtain the N-acetylglucosamine decolorized solution. The decolorized solution was chromatographed by the "717-732" ion exchange resin series system until the conductivity of the discharged liquid was less than 0.5 mS / cm. Then, the desalted solution was concentrated to 500 g / L by multi-effect evaporation, and cooled to 30°C at a cooling rate of 20°C / h at a stirring speed of 250 rpm. At this time, 1.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 10°C for 20 hours before being filtered under reduced pressure. The filtered crystals were washed with 75% ethanol aqueous solution and dried in a blast drying oven at 60°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 76.6%.

[0037] Example 9 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.02% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after the raw material liquid is centrifuged and filtered) of ionic polyacrylamide and 0.18% of sodium alginate were added to the raw material liquid containing N-acetylglucosamine at 70°C to dissolve and flocculate for 10 hours. Subsequently, the flocculated raw material liquid was cross-flow microfiltered through a tubular ceramic membrane to obtain a clarified N-acetylglucosamine sterilized liquid. The sterilized liquid was concentrated under reduced pressure at 50°C to 500g / L, and cooled to 40°C at a cooling rate of 10°C / h at a stirring speed of 500rpm. At this time, 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added, and then filtered under reduced pressure after being kept at 5°C for 5 hours, thereby obtaining N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and filtered to obtain crude sugar 2 and residual mother liquor 2; the residual mother liquor 2 was then concentrated and recrystallized, and filtered to obtain crude sugar 3 and residual mother liquor 3. Then, the N-acetylglucosamine crude sugar obtained by three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 500 g / L. 1.5% activated carbon was added to the solution, and after decolorization for 0.5 h at 50°C and pH 4, it was filtered under reduced pressure to obtain N-acetylglucosamine decolorized liquid. The decolorized liquid was chromatographed by a "732-717" ion exchange resin series system until the conductivity of the discharge liquid was less than 0.1 mS / cm. Then, the desalted liquid was concentrated to 600 g / L by a reverse osmosis system, and cooled to 20°C at a cooling rate of 10°C / h at a stirring speed of 500 rpm. At this time, 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 3°C ​​for 5 hours before being filtered under reduced pressure. The filtered crystals were washed with 90% ethanol aqueous solution and dried in a forced air drying oven at 80°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 78.2%.

[0038] Example 10 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.3% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after the raw material liquid is centrifuged and filtered) of lignin sulfonate was added to the raw material liquid containing N-acetylglucosamine at 40°C to dissolve and flocculate for 10 hours. Subsequently, the flocculated raw material liquid was cross-flow microfiltered through a tubular ceramic membrane to obtain a clarified N-acetylglucosamine sterilized liquid. The sterilized liquid was concentrated to 300 g / L by reduced pressure evaporation at 50°C, and 80% of the mass of the concentrated liquid was slowly added to ethanol at 50°C, and cooled to 20°C at a cooling rate of 1°C / h at a stirring speed of 100 rpm. At this time, 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added, and then vacuum filtered after keeping at 5°C for 5 hours to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and filtered to obtain crude sugar 2 and residual mother liquor 2; the residual mother liquor 2 was then concentrated and recrystallized, and filtered to obtain crude sugar 3 and residual mother liquor 3. Then, the N-acetylglucosamine crude sugar obtained by three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 300 g / L. 1.5% activated carbon was added to the solution, and after decolorization at 50°C and pH 4 for 2.5 hours, it was filtered under reduced pressure at 50°C to obtain N-acetylglucosamine decolorized liquid. The decolorized liquid was chromatographed by a simulated moving bed system of "D150-732" ion exchange resin until the conductivity of the discharge liquid was less than 0.1 mS / cm. Then, the desalted liquid was concentrated under reduced pressure at 50°C to 400 g / L, and cooled to 20°C at a cooling rate of 5°C / h at a stirring speed of 50 rpm. At this time, 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 3°C ​​for 5 hours before being filtered under reduced pressure. The filtered crystals were washed with a 60% ethanol aqueous solution and dried in a blast drying oven at 40°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 87.2%.

[0039] Embodiment 11 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. 0.3% of the chitin of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after the raw material liquid is centrifuged and filtered) was added to the raw material liquid containing N-acetylglucosamine at 70°C to dissolve and flocculate for 10 hours. Subsequently, the flocculated raw material liquid was cross-flow microfiltered through a tubular ceramic membrane to obtain a clarified N-acetylglucosamine sterilization liquid. The sterilization liquid was concentrated to 500g / L by reduced pressure evaporation at 50°C, and 40% of the mass of the concentrated liquid methanol was slowly added at 50°C, and cooled to 40°C at a cooling rate of 10°C / h at a stirring speed of 500rpm. At this time, 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added, and then kept at 10°C for 10 hours and then filtered under reduced pressure to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and filtered to obtain crude sugar 2 and residual mother liquor 2; the residual mother liquor 2 was then concentrated and recrystallized, and filtered to obtain crude sugar 3 and residual mother liquor 3. Then, the N-acetylglucosamine crude sugar obtained by three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 500 g / L. 1.5% activated carbon was added to the solution, and after decolorization for 5 hours at 35°C and pH 4, it was filtered under reduced pressure at 50°C to obtain N-acetylglucosamine decolorized liquid. The decolorized liquid was chromatographed by a simulated moving bed system of "D151-732" ion exchange resin until the conductivity of the discharge liquid was less than 0.5 mS / cm. Then, the desalted liquid was concentrated under reduced pressure at 50°C to 600 g / L, and cooled to 20°C at a cooling rate of 20°C / h at a stirring speed of 500 rpm. At this time, 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 10°C for 10 hours before being filtered under reduced pressure. The filtered crystals were washed with 90% ethanol aqueous solution and dried in a forced air drying oven at 80°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 85.3%.

[0040] Example 12 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. Take 0.3% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after centrifugation and filtration of the raw material liquid) of ionic polyacrylamide, add it to the raw material liquid containing N-acetylglucosamine at 40°C, dissolve it and flocculate it for 10 hours. Subsequently, the flocculated raw material liquid is cross-flow microfiltered through a tubular ceramic membrane to obtain a clear N-acetylglucosamine sterilization liquid. Evaporate under reduced pressure at 50°C to concentrate the sterilization liquid to 300g / L, and slowly add 80% of the mass of the concentrated liquid isopropanol at 50°C, and cool to 20°C at a cooling rate of 1°C / h at a stirring speed of 100rpm. At this time, add 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine), and then keep it at 5°C for 5h and then filter it under reduced pressure to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and the crude sugar 2 and the residual mother liquor 2 were obtained by filtration; the residual mother liquor 2 was then concentrated and recrystallized, and the crude sugar 3 and the residual mother liquor 3 were obtained by filtration. Then, the N-acetylglucosamine crude sugar obtained by the three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 300 g / L. 1.5% activated carbon was added to the solution, and after decolorization for 2.5 hours at 35°C and pH 4, it was filtered under reduced pressure at 50°C to obtain the N-acetylglucosamine decolorized solution. The decolorized solution was chromatographed by a simulated moving bed system until the conductivity of the discharge liquid was less than 0.1 mS / cm. Then, the desalted solution was concentrated to 400 g / L under reduced pressure at 50°C, and cooled to 20°C at a cooling rate of 5°C / h at a stirring speed of 50 rpm. At this time, 0.5% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 3°C ​​for 5 hours before being filtered under reduced pressure. The filtered crystals were washed with a 60% ethanol aqueous solution and dried in a blast drying oven at 40°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 84.8%.

[0041] Example 13 The fermentation liquid obtained by fermentation with the same strain as in Example 1 was used as the raw material liquid of this example. Take 0.3% of the dry weight of the biomass in the raw material liquid (the dry weight is the weight of the dried solid obtained after centrifugation and filtration of the raw material liquid) of ionic polyacrylamide, add it to the raw material liquid containing N-acetylglucosamine at 70°C, dissolve it and flocculate it for 10 hours. Subsequently, the flocculated raw material liquid is cross-flow microfiltered through a tubular ceramic membrane to obtain a clarified N-acetylglucosamine sterilization liquid. Evaporate under reduced pressure at 50°C to concentrate the sterilization liquid to 500g / L, and slowly add 40% of the mass of the concentrated liquid ethylene glycol at 50°C, and cool it to 40°C at a cooling rate of 10°C / h at a stirring speed of 500rpm. At this time, add 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine), and then keep it at 10°C for 10 hours and then filter it under reduced pressure to obtain N-acetylglucosamine crude sugar 1 and residual mother liquor 1. The residual mother liquor 1 was concentrated and recrystallized, and the crude sugar 2 and the residual mother liquor 2 were obtained by filtration; the residual mother liquor 2 was then concentrated and recrystallized, and the crude sugar 3 and the residual mother liquor 3 were obtained by filtration. Then, the N-acetylglucosamine crude sugar obtained by the three crystallizations was re-dissolved with pure water to prepare an N-acetylglucosamine aqueous solution with a concentration of 500 g / L. 1.5% activated carbon was added to the solution, and after decolorization at 50°C and pH 4 for 2.5 hours, it was filtered under reduced pressure at 50°C to obtain the N-acetylglucosamine decolorized liquid. The decolorized liquid was chromatographed by a continuous moving bed system until the conductivity of the discharge liquid was less than 0.5 mS / cm. Then, the desalted liquid was concentrated under reduced pressure at 50°C to 600 g / L, and cooled to 20°C at a cooling rate of 20°C / h at a stirring speed of 500 rpm. At this time, 3% of the total amount of N-acetylglucosamine in the solution (99.9% N-acetylglucosamine) was added again, and the mixture was kept at 10°C for 10 hours before being filtered under reduced pressure. The crystals obtained by filtration were washed with 90% ethanol aqueous solution and dried in a forced air drying oven at 80°C to finally obtain the N-acetylglucosamine product. After high performance liquid chromatography (HPLC) detection, the purity of the N-acetylglucosamine product exceeded 99.5%, and the total yield of the entire process route was 84.2%.

[0042] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for separating and purifying N-acetylglucosamine with high purity and high yield, characterized in that: The following steps are involved: (1) adding a flocculant to a raw material solution containing N-acetylglucosamine for flocculation; the amount of the flocculant added is 0.1-0.6% of the dry weight of the biomass in the raw material solution; the flocculation temperature is 40-70° C.; and the flocculation time is 1-10 hours; (2) performing solid-liquid separation on the solution after flocculation in step (1) to obtain a clarified N-acetylglucosamine sterilization solution and solid waste; (3) concentrating the clarified N-acetylglucosamine sterilized liquid obtained in step (2) and cooling it for crystallization, and performing solid-liquid separation after crystallization to obtain N-acetylglucosamine crude sugar and residual mother liquor; (4) using pure water to redissolve the N-acetylglucosamine crude sugar obtained in step (3) to obtain an N-acetylglucosamine aqueous solution with a concentration of 30-50%, and using an adsorbent to decolorize the N-acetylglucosamine aqueous solution at 20-50° C. and a pH of 4-10 for 0.5-5 h, and after the decolorization, performing solid-liquid separation to obtain an N-acetylglucosamine decolorized solution and an adsorbent; (5) desalting the N-acetylglucosamine decolorizing solution obtained in step (4) to obtain an N-acetylglucosamine desalted solution, wherein the final conductivity of the desalted solution is 0.1-0.5 mS / cm; the desalting method is ion exchange chromatography; (6) Concentrating the N-acetylglucosamine desalted liquid obtained in step (5) and cooling it for crystallization, and performing solid-liquid separation after crystallization to obtain N-acetylglucosamine crystals and residual clear liquid; the residual clear liquid is refluxed to step (5); the N-acetylglucosamine crystals are washed with an ethanol aqueous solution to obtain high-purity N-acetylglucosamine crystals; (7) Drying the high-purity N-acetylglucosamine crystals obtained in step (6); the drying method is air drying.

2. The method for separating and purifying N-acetylglucosamine with high purity and high yield according to claim 1, characterized in that: The raw material liquid in step (1) is the fermentation liquid obtained by microbial fermentation.

3. The method for separating and purifying N-acetylglucosamine with high purity and high yield according to claim 1, characterized in that: The flocculant in step (1) is at least one of aluminum sulfate, ferric chloride, ferrous sulfate, ferric sulfate, polyferric sulfate, polyaluminum chloride, gelatin, sodium alginate, polyacrylamide, lignin sulfonate, chitin and their derivatives; when two or more flocculants are used, the proportion of each component flocculant is not less than 10%.

4. The method for separating and purifying N-acetylglucosamine with high purity and high yield according to claim 1, characterized in that: The solid-liquid separation method in step (2) is microfiltration or centrifugation; the microfiltration component is tubular, plate-and-frame or roll-type; the microfiltration membrane material is an organic polymer membrane or a ceramic membrane; the microfiltration method is cross-flow filtration; and the centrifugation uses a disc centrifuge.

5. The method for separating and purifying N-acetylglucosamine with high purity and high yield according to claim 1, characterized in that: The concentration method in step (3) is vacuum concentration, flash concentration or multiple-effect evaporation concentration; the solid-liquid separation method is vacuum filtration or centrifugation; the residual mother liquor obtained by batch separation is recrystallized twice, and the solid-liquid separation operation is repeated to finally obtain three-crystallized raw sugar.

6. The method for separating and purifying N-acetylglucosamine with high purity and high yield according to claim 1, characterized in that: The concentration and crystallization method in step (3) is (a) or (b): (a) The clarified N-acetylglucosamine sterilized solution is concentrated at 45-80°C to obtain an N-acetylglucosamine concentrated solution with a concentration of 30-50%, and then crystallized; the cooling rate of the crystallization is 1-10°C / h; the crystallization stirring rate is 0-500rpm; when the temperature is lowered to 20-40°C, 0.5-3% of the total amount of N-acetylglucosamine in the solution is added with seeds; the seeds are 99.9% N-acetylglucosamine; the crystallization temperature is maintained at 5-10°C; the crystallization time is maintained at 5-20h; (b) The clarified N-acetylglucosamine sterilizing solution is concentrated at 45-80°C to obtain an N-acetylglucosamine concentrated solution with a concentration of 30-50%, and a food safety solvent with a mass fraction of 5-50% is added to perform crystallization; the solvent is anhydrous ethanol, anhydrous methanol, ethylene glycol, and isopropanol; the cooling rate of the crystallization is 1-10°C / h; the crystallization stirring rate is 0-500rpm; when the temperature is lowered to 20-40°C, 0.5-3% of the total amount of N-acetylglucosamine in the solution is added with seed crystals; the seed crystals are 99.9% N-acetylglucosamine; the crystallization temperature is maintained at 5-10°C; and the crystallization time is maintained at 5-20h.

7. The method for separating and purifying N-acetylglucosamine with high purity and high yield according to claim 1, characterized in that: The adsorbent in step (4) is activated carbon, macroporous decolorizing resin, diatomaceous earth or bentonite; the amount of activated carbon added is 0.5-2% of the mass of the N-acetylglucosamine aqueous solution; the amount of macroporous decolorizing resin added is 5-20% of the mass of the N-acetylglucosamine aqueous solution; the amount of diatomaceous earth added is 2-10% of the mass of the N-acetylglucosamine aqueous solution; the amount of bentonite added is 2-10% of the mass of the N-acetylglucosamine aqueous solution.

8. The method for separating and purifying N-acetylglucosamine with high purity and high yield according to claim 1, characterized in that: The ion exchange chromatography in step (5) uses anion exchange resin and cation exchange resin to perform bipolar resin chromatography desalting.

9. The method for separating and purifying N-acetylglucosamine with high purity and high yield according to claim 1, characterized in that: In step (6), the N-acetylglucosamine desalted liquid is concentrated to obtain an N-acetylglucosamine concentrated liquid with a concentration of 40-70%; the solid-liquid separation method is vacuum filtration; the separated N-acetylglucosamine crystals are washed with an ethanol aqueous solution with a mass fraction of 60-90%; the concentration method is at least one of vacuum evaporation concentration, flash concentration, multi-effect evaporation concentration or reverse osmosis membrane concentration.

10. The method for separating and purifying N-acetylglucosamine with high purity and high yield according to claim 1, characterized in that: In step (6), the N-acetylglucosamine desalted solution is concentrated at 45-80°C; the crystallization cooling rate is 5-20°C / h; the crystallization stirring rate is 0-400rpm; when the temperature is cooled to 20-40°C, 0.5-3% of the total amount of N-acetylglucosamine in the solution is added with seeds; the seeds are 99.9% N-acetylglucosamine; the crystallization temperature is maintained at 3-10°C; and the crystallization time is maintained at 5-20h.