A process for preparing glucosamine using N-acetylglucosamine fermentation broth

Abstract

The present invention discloses a process for preparing glucosamine from N-acetylglucosamine fermentation broth, which comprises the steps of: 1. Mixing a Ti 4+ source and an Fe 3+ source, adding an alkali solution thereto for precipitation; then separating out the solid product, washing and drying it to obtain a precursor; impregnating the precursor with an ammonium persulfate solution, drying the obtained precursor and then calcining it to obtain a modified solid superacid; 2. Adding the modified solid superacid to the N-acetylglucosamine fermentation broth, carrying out hydrolysis under heating conditions, irradiating the reaction system with a light source after completion, and carrying out solid-liquid separation after completion to obtain a hydrolysis solution; 3. Concentrating the hydrolysis solution, carrying out alcohol precipitation, then separating out the solid product and drying it to obtain a glucosamine product. The process of the present invention not only overcomes the above problems brought about by hydrolysis with concentrated acid solution, but also can use the solid superacid to decolorize the hydrolysis solution and improve the purity of the obtained glucosamine product.

Description

Technical Field

[0001] The present invention relates to the technical field of glucosamine, and particularly to a process for preparing glucosamine by using an N-acetylglucosamine fermentation broth. Background Art

[0002] Glucosamine is an amino monosaccharide in which the hydroxyl group at the 2-position carbon of glucose is replaced by an amino group. Glucosamine is one of the important monomers that make up polysaccharides such as hyaluronic acid, heparin, and keratan sulfate in organisms. At the same time, it is also a precursor substance for the synthesis of human milk oligosaccharides, neuraminic acid, and chitosan oligosaccharides, and can maintain the normal physiological functions of organisms. In recent years, glucosamine and its derivatives (such as sulfates, hydrochlorides, etc.) have been widely used in the fields of food, medicine, and cosmetics, and the demand has been increasing year by year, with broad market application prospects.

[0003] Currently, the preparation processes of glucosamine mainly include chemical extraction methods and microbial fermentation methods. The chemical extraction method uses shrimp and crab shells as raw materials, extracts chitin or chitosan from them, and then obtains glucosamine through hydrochloric acid hydrolysis. However, this glucosamine has a fishy smell and is likely to cause allergies in people sensitive to seafood. In addition, since heavy metals in shrimp and crab shells are likely to exceed the standard, the glucosamine prepared by the above chemical extraction method is also likely to have the problem of heavy metal exceeding the standard. Relatively speaking, the glucosamine prepared by the microbial fermentation method does not have the above problems. This method first uses an engineered bacterium to produce N-acetylglucosamine, which forms glucosamine after concentrated acid hydrolysis. However, the use of concentrated acid brings more pollution, generates a large amount of waste liquid, and the concentrated acid seriously corrodes the equipment. Summary of the Invention

[0004] In view of the above problems, the present invention provides a process for preparing glucosamine by using an N-acetylglucosamine fermentation broth, which uses a modified solid superacid to replace the concentrated acid solution for hydrolysis, not only overcomes the above problems brought by the concentrated acid solution, but also can use the solid superacid to decolorize the hydrolysis solution and improve the purity of the obtained glucosamine product. Specifically, the technical solution of the present invention is as follows.

[0005] A process for preparing glucosamine by using an N-acetylglucosamine fermentation broth, comprising the steps of:

[0006] (1) Preparation of the modified solid superacid: Mix the Ti 4+ source and the Fe 3+ source evenly, then add an alkali solution while stirring, and let it stand after the precipitation is complete. After completion, separate the solid product, wash and dry it to obtain the precursor. Impregnate the precursor with an ammonium persulfate solution, and after completion, dry the obtained precursor and then calcine it to obtain the modified solid superacid (S2O8 2- / (TiO2-Fe2O3).

[0007] (2) Add the modified solid superacid to the fermentation broth of N-acetylglucosamine, and then carry out a hydrolysis reaction under heating conditions. After completion, irradiate the reaction system with a light source, and then carry out solid-liquid separation to obtain the hydrolyzate.

[0008] (3) Concentrate the hydrolyzate, carry out alcohol precipitation, then separate out the solid product and dry it to obtain the glucosamine product.

[0009] Further, in step (1), the molar ratio of the Ti 4+ and Fe 3+ is 3-5:1-2. Optionally, the Ti 4+ source includes any one of titanium chloride, titanium nitrate, etc. The Fe 3+ source includes any one of ferric chloride, ferric nitrate, etc.

[0010] Further, in step (1), add the alkali solution until the pH of the reaction system is between 9 and 11, and then let it stand for 20-24 hours. Optionally, the alkali solution includes any one of ammonia water, sodium hydroxide, etc.

[0011] Further, in step (1), wash the solid product with clear water to remove residual soluble ions, and then dry it at 95-110 °C for 10-12 hours to obtain the precursor.

[0012] Further, in step (1), the concentration of the ammonium persulfate solution is 0.4-0.7 mol / L. The amount of the ammonium persulfate solution used is sufficient to fully impregnate the precursor.

[0013] Further, in step (1), the impregnation time is 10-14 hours to fully introduce sulfate radicals (S2O8 2- ) into the precursor. After completion, dry the obtained precursor at 95-110 °C for 1-2 hours.

[0014] Further, in step (1), the calcination temperature is 470-550 °C, and the calcination time is 2-3.5 hours to form the modified solid superacid (S2O8 2- / TiO2-Fe2O3).

[0015] Further, in step (2), the mass ratio of N-acetylglucosamine to the modified solid superacid in the fermentation broth is 10:4-5.5.

[0016] Further, in step (2), the heating temperature is 40-60 °C, and the hydrolysis reaction time is 2-4 hours.

[0017] Further, in step (2), the power of the light source is 60 - 100 W, and the irradiation time is 45 - 70 min.

[0018] Further, in step (3), the hydrolysis solution is concentrated to 40 - 55% of the initial volume by heating. Then, ethanol with a volume 3 - 5 times that of the obtained concentrated solution is added for alcohol precipitation so that glucosamine in the concentrated solution crystallizes out.

[0019] Further, in step (3), the drying methods include freeze - drying, vacuum drying, etc.

[0020] Compared with the prior art, the present invention has the following beneficial technical effects: The present invention uses a modified solid superacid to hydrolyze N - acetylglucosamine in the fermentation broth instead of concentrated hydrochloric acid, converting it into glucosamine. This not only overcomes the above - mentioned problems brought by concentrated acid solution, but also can use the solid superacid to decolorize the hydrolysis solution and improve the purity of the obtained glucosamine product. The reason is as follows: First, the modified solid superacid has highly active acid active centers S2O8 2- , under its catalytic action, N - acetylglucosamine is hydrolyzed into glucosamine. This method not only avoids the pollution problem caused by using concentrated hydrochloric acid, but also since the modified solid superacid can be easily separated from the obtained hydrolysis solution by solid - liquid separation, there is no need to remove the residual concentrated hydrochloric acid by adding alkali solution for neutralization, which in turn causes the obtained glucosamine to actually be glucosamine hydrochloride, and the large amount of chloride ions it contains has some adverse effects. However, when the present invention uses the modified solid superacid for the hydrolysis of N - acetylglucosamine, chloride ions will not be introduced, thus well overcoming the above - mentioned problems. In addition, the modified solid superacid prepared in the present invention uses TiO2 as a carrier and is doped with Fe2O3. It not only acts as a promoter to further enhance the catalytic efficiency and improve the hydrolysis efficiency of N - acetylglucosamine. Moreover, Fe2O3 extends the absorption edge band of TiO2 to the visible light region, so that the modified solid superacid of the present invention can use visible light for photocatalytic degradation and decolorization. During the illumination process, the active oxygen generated by the modified solid superacid degrades the pigment in the hydrolysis solution into water and carbon dioxide, thereby improving the purity of the obtained glucosamine product. Specific Embodiments

[0021] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present invention. The present invention will be further described through specific examples. Example 1

[0022] The process for preparing glucosamine using N - acetylglucosamine fermentation broth in this example includes the steps:

[0023] 1. Preparation of modified solid superacid:

[0024] Mix the TiCl4 solution and FeCl3 solution evenly by stirring according to the molar ratio of Ti 4+ and Fe 3+ being 4:1. Then add concentrated ammonia water to the reaction system under rapid stirring until the pH of the reaction system reaches 10, and then let it stand for 24 hours. After completion, centrifuge to separate the solid product, wash the solid product three times with clear water, place it in an oven, and dry it at 100 °C for 12 hours to obtain the precursor.

[0025] Immerse the precursor in a 0.45 mol / L ammonium persulfate solution for 12 hours. After completion, place the obtained precursor in an oven and dry it at 100 °C for 1.5 hours. Then place the obtained precursor in a muffle furnace and calcine it at 500 °C for 3 hours to obtain the modified solid superacid.

[0026] 2. According to the mass ratio of N - acetylglucosamine to the modified solid superacid in the fermentation broth being 10:4.5, add the modified solid superacid prepared in this example to the fermentation broth. Then heat it in a water bath to 50 °C and keep it warm for 3 hours for hydrolysis reaction. After completion, irradiate the reaction system with a 70 W xenon lamp for 60 min. After completion, centrifuge to separate the modified solid superacid, and collect the liquid phase to obtain the hydrolyzate.

[0027] 3. Concentrate the hydrolyzate to 50% of the initial volume by heating in a water bath. Then add anhydrous ethanol four times the volume of the obtained concentrated liquid, stir evenly, and let it stand for 40 min for alcohol precipitation. After completion, centrifuge to separate the solid product, and vacuum - dry it at 80 °C for 1 hour to obtain the glucosamine product. Use spectrophotometry to test the purity of the glucosamine product in this example, and the result is 92.33%. Example 2

[0028] The process for preparing glucosamine from N - acetylglucosamine fermentation broth in this example includes the steps:

[0029] 1. Preparation of modified solid superacid:

[0030] Mix the TiCl4 solution and FeCl3 solution evenly by stirring according to the molar ratio of Ti 4+ and Fe 3+ being 3:1. Then add sodium hydroxide to the reaction system under rapid stirring until the pH of the reaction system reaches 11, and then let it stand for 22 hours. After completion, centrifuge to separate the solid product, wash the solid product three times with clear water, place it in an oven, and dry it at 95 °C for 11 hours to obtain the precursor.

[0031] The precursor is immersed in an ammonium persulfate solution with a concentration of 0.4 mol / L for 10 hours. After completion, the obtained precursor is placed in an oven and dried at 95 °C for 2 hours. Then, the obtained precursor is placed in a muffle furnace and calcined at 550 °C for 2 hours to obtain the modified solid superacid.

[0032] 2. According to the mass ratio of N-acetylglucosamine to the modified solid superacid in the fermentation broth of 10:5.5, the modified solid superacid prepared in this example is added to the fermentation broth. Then, it is heated in a water bath to 60 °C and kept warm for 2 hours for hydrolysis reaction. After completion, the reaction system is irradiated with a 60 W xenon lamp for 70 min. After completion, the modified solid superacid is separated by centrifugation, and the liquid phase is collected to obtain the hydrolyzate.

[0033] 3. The hydrolyzate is concentrated to 55% of the initial volume by heating in a water bath. Then, anhydrous ethanol with a volume 5 times that of the obtained concentrated solution is added, and after stirring evenly, it is left standing for 45 min for alcohol precipitation. After completion, the solid product is separated by centrifugation and vacuum dried at 80 °C for 1 hour to obtain the glucosamine product. The purity of the glucosamine product in this example is measured by spectrophotometry, and the result is 94.07%. Example 3

[0034] The process for preparing glucosamine from the N-acetylglucosamine fermentation broth described in this example includes the steps:

[0035] 1. Preparation of the modified solid superacid:

[0036] The Ti(NO3)4 solution and the Fe(NO3)3 solution are mixed evenly according to the molar ratio of Ti 4+ , Fe 3+ of 5:2. Then, concentrated ammonia water is added under rapid stirring until the pH of the reaction system reaches 9, and then it is left standing for 20 hours. After completion, the solid product is separated by centrifugation, washed three times with clear water, placed in an oven, and dried at 110 °C for 10 hours to obtain the precursor.

[0037] The precursor is immersed in an ammonium persulfate solution with a concentration of 0.7 mol / L for 14 hours. After completion, the obtained precursor is placed in an oven and dried at 110 °C for 1 hour. Then, the obtained precursor is placed in a muffle furnace and calcined at 470 °C for 3.5 hours to obtain the modified solid superacid.

[0038] 2. Add the modified solid superacid prepared in this example to the fermentation broth according to the mass ratio of N-acetylglucosamine to the modified solid superacid in the fermentation broth of 10:4. Then, heat in a water bath to 40 °C and keep warm for 4 hours to carry out the hydrolysis reaction. After completion, irradiate the reaction system with a 100 W xenon lamp for 45 min. After completion, centrifuge to separate the modified solid superacid, and collect the liquid phase to obtain the hydrolysis solution.

[0039] 3. Concentrate the hydrolysis solution to 40% of the initial volume by heating in a water bath. Then, add anhydrous ethanol three times the volume of the obtained concentrated solution, stir evenly, and let stand for 35 min for alcohol precipitation. After completion, centrifuge to separate the solid product, and freeze-dry it to obtain the glucosamine product. The purity of the glucosamine product in this example was tested by spectrophotometry, and the result was 94.67%. Example 4

[0040] The process for preparing glucosamine from N-acetylglucosamine fermentation broth described in this example includes the steps:

[0041] 1. Preparation of the modified solid superacid:

[0042] Add concentrated ammonia water to the TiCl4 solution under rapid stirring until the pH of the reaction system is 10, and then let stand for 24 hours. After completion, centrifuge to separate the solid product, wash the solid product three times with clear water, place it in an oven, and dry it at 100 °C for 12 hours to obtain the precursor.

[0043] Immerse the precursor in a 0.45 mol / L ammonium persulfate solution for 12 hours. After completion, place the obtained precursor in an oven and dry it at 100 °C for 1.5 hours. Then, place the obtained precursor in a muffle furnace and calcine it at 500 °C for 3 hours to obtain the modified solid superacid.

[0044] 2. Add the modified solid superacid prepared in this example to the fermentation broth according to the mass ratio of N-acetylglucosamine to the modified solid superacid in the fermentation broth of 10:4.5. Then, heat in a water bath to 50 °C and keep warm for 3 hours to carry out the hydrolysis reaction. After completion, irradiate the reaction system with a 70 W xenon lamp for 60 min. After completion, centrifuge to separate the modified solid superacid, and collect the liquid phase to obtain the hydrolysis solution.

[0045] 3. Concentrate the hydrolysis solution to 50% of the initial volume by heating in a water bath. Then, add anhydrous ethanol four times the volume of the obtained concentrated solution, stir evenly, and let stand for 40 min for alcohol precipitation. After completion, centrifuge to separate the solid product, and dry it in vacuum at 80 °C for 1 hour to obtain the glucosamine product. The purity of the glucosamine product in this example was tested by spectrophotometry, and the result was 86.52%. Example 5

[0046] The process for preparing glucosamine from N-acetylglucosamine fermentation broth described in this example includes the following steps:

[0047] 1. Preparation of modified solid superacid:

[0048] Mix the TiCl4 solution and FeCl3 solution evenly according to the molar ratio of Ti 4+ 、Fe 3+ being 3:1. Then add sodium hydroxide to the reaction system under rapid stirring until the pH = 11, and then let it stand for 22 hours. After completion, centrifuge to separate the solid product, wash the solid product three times with clear water, place it in an oven, and dry it at 95°C for 11 hours to obtain the precursor.

[0049] Place the precursor in an oven and dry it at 95°C for 2 hours. Then place the obtained precursor in a muffle furnace and calcine it at 550°C for 2 hours to obtain the modified solid superacid.

[0050] 2. Add the modified solid superacid prepared in this example to the fermentation broth according to the mass ratio of N-acetylglucosamine to the modified solid superacid in the fermentation broth being 10:5.5. Then heat it in a water bath to 60°C and keep it warm for 2 hours for hydrolysis reaction. After completion, irradiate the reaction system with a 60W xenon lamp for 70 minutes. After completion, centrifuge to separate the modified solid superacid, and collect the liquid phase to obtain the hydrolyzate.

[0051] 3. Concentrate the hydrolyzate to 55% of the initial volume by heating in a water bath. Then add anhydrous ethanol 5 times the volume of the obtained concentrated solution, stir evenly, and let it stand for 45 minutes for alcohol precipitation. After completion, centrifuge to separate the solid product, and vacuum dry it at 80°C for 1 hour to obtain the glucosamine product. Use spectrophotometry to test the purity of the glucosamine product described in this example, and the result is 31.26%. Example 6

[0052] The process for preparing glucosamine from N-acetylglucosamine fermentation broth described in this example includes the following steps:

[0053] 1. Preparation of modified solid superacid:

[0054] Add concentrated ammonia water to the Fe(NO3)3 solution under rapid stirring until the pH = 9 of the reaction system, and then let it stand for 20 hours. After completion, centrifuge to separate the solid product, wash the solid product three times with clear water, place it in an oven, and dry it at 110°C for 10 hours to obtain the precursor.

[0055] The precursor is immersed in an ammonium persulfate solution with a concentration of 0.7 mol / L for 14 hours. After completion, the obtained precursor is placed in an oven and dried at 110 °C for 1 hour. Then, the obtained precursor is calcined in a muffle furnace at 470 °C for 3.5 hours to obtain the modified solid superacid.

[0056] 2. According to the mass ratio of N-acetylglucosamine to the modified solid superacid in the fermentation broth of 10:4, the modified solid superacid prepared in this example is added to the fermentation broth. Then, it is heated in a water bath to 40 °C and kept warm for 4 hours to carry out the hydrolysis reaction. After completion, the reaction system is irradiated with a 100 W xenon lamp for 45 min. After completion, the modified solid superacid is separated by centrifugation, and the liquid phase is collected to obtain the hydrolysis solution.

[0057] 3. The hydrolysis solution is concentrated to 40% of the initial volume by heating in a water bath. Then, anhydrous ethanol with a volume three times that of the obtained concentrated solution is added, and after stirring evenly, it is left to stand for 35 min for alcohol precipitation. After completion, the solid product is separated by centrifugation and freeze-dried to obtain the glucosamine product. The purity of the glucosamine product described in this example is measured by spectrophotometry, and the result is 89.84%.

[0058] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A process for preparing glucosamine using N - acetylglucosamine fermentation broth, characterized in that: It includes the steps: (1) Preparation of modified solid superacid: Mix the Ti 4+ source and the Fe 3+ source evenly, then add the alkali solution while stirring, and let it stand after the precipitation is complete; After completion, the solid product is separated out, washed and dried to obtain the precursor; the precursor is impregnated with an ammonium persulfate solution, and after completion, the obtained precursor is dried and then calcined to obtain the modified solid superacid; (2) Add the modified solid superacid to the fermentation broth of N-acetylglucosamine, and then carry out a hydrolysis reaction under heating conditions. After completion, the reaction system is irradiated with a light source, and after completion, solid-liquid separation is carried out to obtain the hydrolysis solution; (3) Concentrate the hydrolysis solution and then carry out alcohol precipitation, and then separate out the solid product and dry it to obtain the glucosamine product; In step (1), the molar ratio of Ti 4+ to Fe 3+ is 3 to 5: 1 to 2; the Ti 4+ source includes any one of titanium chloride and titanium nitrate; the Fe 3+ source includes any one of ferric chloride and ferric nitrate; In step (1), add the alkali solution until the pH of the reaction system is between 9 and 11; In step (1), the concentration of the ammonium persulfate solution is 0.4 - 0.7 mol / L; In step (1), the calcination temperature is 470 - 550 °C, and the calcination time is 2 - 3.5 hours.

2. The process for preparing glucosamine from N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (1), the standing time is 20 - 24 hours.

3. The process for preparing glucosamine from N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (1), the alkali solution includes any one of ammonia water and sodium hydroxide.

4. The process for preparing glucosamine using N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (1), the solid product is washed with clear water and then dried at 95 - 110 °C for 10 - 12 hours to obtain the precursor.

5. The process for preparing glucosamine using N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (1), the impregnation time is 10 - 14 hours; after completion, the obtained precursor can be dried at 95 - 110 °C for 1 - 2 hours.

6. The process for preparing glucosamine using N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (2), the mass ratio of N-acetylglucosamine to the modified solid superacid in the fermentation broth is 10:4 - 5.

5.

7. The process for preparing glucosamine using N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (2), the heating temperature is 40 - 60 °C, and the hydrolysis reaction time is 2 - 4 hours.

8. The process for preparing glucosamine using N - acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (2), the power of the light source is 60 - 100 W, and the irradiation time is 45 - 70 min.

9. The process for preparing glucosamine from N-acetylglucosamine fermentation broth according to any one of claims 1-8, characterized in that: In step (3), heat to concentrate the hydrolysis solution to 40 - 55% of the initial volume; then add ethanol 3 - 5 times the volume of the obtained concentrated solution for alcohol precipitation.

10. The process for preparing glucosamine using N - acetylglucosamine fermentation broth according to any one of claims 1 - 8, characterized in that: In step (3), the drying method includes freeze-drying or vacuum drying.