Method for separating and extracting tryptophan from fermentation liquor

Abstract

The invention provides a method for separating and extracting tryptophan from fermentation liquor, and belongs to the technical field of tryptophan extraction. The extraction method comprises the following steps: adding formic acid into tryptophan fermentation liquor, adjusting the pH value to 5.80-5.90, then reducing the temperature to 5.8-6.2 DEG C, carrying out heat preservation and standing to obtain crystallized mixed liquor, carrying out centrifugal treatment to obtain precipitated coarse crystals, putting the precipitated coarse crystals into deionized water, fully and uniformly dissolving the precipitated coarse crystals, adding porous activated carbon, stirring and centrifuging to obtain decolored liquor; adjusting the pH value of the decoloring solution to 3.0-3.4 by using formic acid, introducing the decoloring solution into an ion exchange column, filling cation exchange resin into the ion exchange column, eluting by using an ammonia water solution after adsorption is completed to obtain a tryptophan mixed solution, crystallizing and drying to obtain a tryptophan product; the tryptophan product obtained by the method is high in purity and high in yield, and the regeneration capacity of the cation exchange resin is high.

Description

Technical Field

[0001] The invention belongs to the technical field of tryptophan extraction, and in particular relates to a method for separating and extracting tryptophan from fermentation liquid. Background Art

[0002] Tryptophan, also known as β-indolylalanine, has a solubility of 1.14 g / L in water at room temperature. It is relatively stable in alkali solution and easily decomposed in strong acid. It is one of the eight essential amino acids in human and animal life activities. It plays an important role in the growth, development and metabolism of humans and animals. It has the functions of regulating protein synthesis, improving immunity, improving sleep quality, and relieving tension. It is widely used in food, feed additives, medicine, agriculture and other fields.

[0003] The production methods of tryptophan mainly include chemical synthesis, enzymatic method and microbial fermentation method; the material source of the chemical synthesis method is limited, and the substrate is expensive; the enzyme method requires a large amount of enzyme and is easily inactivated, and the enzyme is expensive, which increases the production cost; the microbial fermentation method uses glucose as raw material, has the advantages of high acid production, mild reaction conditions, wide raw material sources and low cost, and has become the main technology for producing tryptophan.

[0004] When tryptophan is prepared by microbial fermentation, the fermentation liquid is dark in color, and the protein produced by the bacteria increases the viscosity of the fermentation liquid. The presence of protein and pigment increases the difficulty of extracting tryptophan and directly affects the yield and purity of the product. In the existing preparation process of crude tryptophan, ultrafiltration membrane technology, ion exchange method, or a combination of the two are generally used; First, the ultrafiltration membrane technology is simply used to filter the feed liquid to remove bacteria, proteins, etc., but the membrane resistance increases and the flux decreases during long-term operation. It is also expensive, has a small pore size, is very easy to get clogged, and takes a long time to clean. A large amount of wastewater is also generated during the cleaning process, causing environmental pollution. In addition, the solubility of tryptophan in water is only 1.14g / L, and it is easily retained by the membrane after crystallization, resulting in unnecessary losses.

[0005] Furthermore, CN113527176A discloses a method for extracting and separating tryptophan from a fermentation broth, which includes pretreatment of the fermentation broth - crude extraction with a cation exchange resin - purification with a macroporous adsorption resin - decolorization and desalination - freeze drying. The process omits the ultrafiltration membrane filtration operation, removes proteins and pigments with a cation exchange resin, crudely extracts tryptophan from the fermentation broth, and then further separates and purifies the crude extract with a macroporous resin to obtain tryptophan. This patent simply adopts the ion exchange method, which combines a cation exchange resin with a macroporous resin. The cation exchange resin has poor exchange capacity and selectivity, which also affects the extraction effect of tryptophan. The purity of the obtained tryptophan is low, only 90.3%.

[0006] CN101914054A discloses a new process for separating and extracting L-tryptophan from fermentation broth, comprising pre-treating the fermentation broth, pumping the treated broth into a ceramic microfiltration membrane filter to obtain a tryptophan microfiltrate with bacteria removed and bacterial protein; pumping the tryptophan microfiltrate into an ultrafiltration membrane filter to obtain a tryptophan ultrafiltrate and a concentrated solution containing pigments and proteins; pumping the tryptophan ultrafiltrate into a nanofiltration membrane filter to obtain a tryptophan concentrated solution and a salt-containing permeate; comprehensively utilizing the filtered waste liquid to prepare a protein feed; applying industrial acetic acid to promote low-temperature crystallization, treating the crystallization mother liquor with an ion exchange method, and centrifuging, dehydrating, and drying the crystals to obtain the finished L-tryptophan; This process combines ultrafiltration membrane technology with ion exchange resin, and its equipment cost is relatively high, and the yield of tryptophan is still low, only 85.3%.

[0007] Therefore, providing a method for separating and extracting tryptophan from a fermentation broth, avoiding the use of high-cost equipment such as ultrafiltration membranes, improving the ion exchange performance of cation exchange resins, and increasing the yield and purity of tryptophan are technical problems that need to be solved urgently in the prior art. Summary of the invention

[0008] In order to solve the technical problems existing in the prior art, the present invention provides a method for separating and extracting tryptophan from a fermentation broth, avoiding the use of high-cost equipment such as ultrafiltration membranes, improving the ion exchange performance of cation exchange resins, and improving the yield and purity of tryptophan.

[0009] In view of the above technical problems, the present invention adopts the following technical solutions: A method for separating and extracting tryptophan from a fermentation broth, comprising pretreatment, decolorization, ion exchange and post-treatment steps, specifically as follows: 1. Pre-treatment Formic acid is added to the tryptophan fermentation broth to adjust the pH to 5.80-5.90, and then the temperature is reduced to 5.8-6.2°C at a rate of 1.5-2.5°C / min, and the mixture is kept warm and allowed to stand for 13.5-14.5 hours to obtain a crystallization mixture; the crystallization mixture is centrifuged at a speed of 1500-1600 rpm for 5.0-7.0 minutes to obtain precipitated crude crystals after the centrifugation.

[0010] 2. Decolorization The precipitated crude crystals are put into 18-22 times the mass of deionized water to prepare a crude crystal mixed solution, the crude crystal mixed solution is heated to 65-70°C, after being fully dissolved and uniformly, porous activated carbon is added, and stirred at a stirring speed of 200-220rpm for 18-22min. After stirring, centrifugation is performed at a centrifugal speed of 3900-4100rpm for 8-12min. After centrifugation, a decolorized solution is obtained; The porous activated carbon is 1.8-2.2% of the mass of the mixed liquid; The preparation method of the porous activated carbon is as follows: placing the activated carbon in a hydrogen peroxide solution and stirring it for 1.4-1.6 hours at a stirring temperature of 43-47° C. After the stirring is completed, filtering, washing, and drying, the mixture is kept at 117-123° C. for 1.8-2.2 hours, and then naturally recovered to room temperature to obtain the porous activated carbon; The mass ratio of the activated carbon to the hydrogen peroxide solution is 9.5-10.5:95-105; The mass concentration of the hydrogen peroxide solution is 3.2-3.8%.

[0011] 3. Ion exchange The decolorizing solution is adjusted to a pH of 3.0-3.4 with formic acid, and passed into an ion exchange column filled with a cation exchange resin, and the temperature is controlled at 28-32° C., and the flow rate is 1.5-1.7 BV / h. After the adsorption is completed, 1.8-2.2 mol / L ammonia solution is used for elution, and the elution temperature is 54-56° C., and the elution flow rate is 1.8-2.2 BV / h. After the elution is completed, a tryptophan mixed solution is obtained; The preparation method of the cation exchange resin includes copolymerization and sulfonation steps, which are specifically as follows: The copolymerization reaction step comprises: adding deionized water to a reaction container, then adding gelatin and sodium cocoyl methyl taurate and stirring, stirring evenly to obtain a dispersant; mixing styrene, divinylbenzene, white oil and benzoyl peroxide, stirring evenly, adding the mixture to the dispersant, controlling the adding rate to be 1.3-1.7 g / min, adding pre-modified kaolin, and homogenizing the mixture, wherein the homogenization time is 4-6 min, the homogenization pressure is 1.6-2.0 MPa, the homogenization times are 2 times, and after the homogenization is completed, heating the mixture to 78-82° C. at a rate of 0.1-0.3° C. / min, and keeping the temperature for reaction for 6.7-7.3 h. After the reaction is completed, cooling, washing and drying the mixture to obtain copolymer microspheres; The mass ratio of the deionized water, gelatin and sodium cocoyl methyl taurate is 290-310:0.50-0.55:0.14-0.16; The mass ratio of styrene, divinylbenzene, white oil, benzoyl peroxide, dispersant and pre-modified kaolin is 25-30:8.8-9.2:10.6-11.0:0.25-0.30:300-304:8.0-8.5; The preparation method of the pre-modified kaolin is as follows: calcining the kaolin at 345-355° C. for 3.8-4.2 hours, naturally cooling to room temperature, placing the kaolin in a nitric acid solution, raising the temperature to 128-132° C. for reflux reaction, and the reaction time is 1.4-1.6 hours. After the reaction is completed, filtering and washing to obtain acid-washed kaolin; putting the acid-washed kaolin into an ethanol solution, adding polyethylene glycol 400 and sodium ricinoleate, stirring evenly, adding triethylenetetramine, raising the temperature to 56-60° C., keeping warm and stirring for 0.8-1.2 hours, filtering, washing, and drying after the insulation is completed to obtain the pre-modified kaolin; The particle size of the kaolin is 234-245nm; The mass ratio of kaolin to nitric acid solution is 18-22:176-184; The mass concentration of the nitric acid solution is 72-75%; The mass ratio of the acid-washed kaolin, ethanol solution, polyethylene glycol 400, sodium castor oil sulfonate and triethylenetetramine is 17.8-18.2:175-185:1.3-1.5:1.0-1.2:4.1-4.5; The mass concentration of the ethanol solution is 38-42%; The sulfonation step comprises placing the copolymer microspheres in ethylene dichloride, controlling the temperature to 58-62° C., keeping the temperature and stirring for 1.7-2.3 hours, adding concentrated sulfuric acid after the temperature is kept at 108-113° C. at a rate of 0.8-1.2° C. / min, stirring to evaporate the ethylene dichloride, keeping the temperature and reacting for 11.7-12.3 hours, and cooling, filtering, washing and drying after the reaction to obtain the sulfonated copolymer microspheres; The mass volume ratio of the copolymer microspheres, ethylene dichloride and concentrated sulfuric acid is 9.8-10.3 g: 290-310 mL: 490-510 mL; The mass concentration of the concentrated sulfuric acid is 98%.

[0012] 4. Post-processing The tryptophan mixture is placed in a crystallization device for crystallization, the crystallization temperature is 1.8-2.2°C, the pH is controlled to be 5.90-6.0, the crystallization time is 9.5-10.5h, and primary tryptophan crystals are obtained by centrifugation. The primary tryptophan crystals are vacuum dried at a drying temperature of 78-82°C, a drying time of 3.0-3.5h, and a vacuum degree of -0.06 to -0.09MPa. After drying, the mixture is naturally restored to room temperature to obtain a tryptophan product.

[0013] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The present invention directly performs low-temperature crystallization on the tryptophan fermentation broth, and through centrifugation, most of the bacterial cells, proteins, pigments and other components in the fermentation broth are left in the mother liquor, so as to achieve separation from tryptophan, avoid filtration by ultrafiltration membrane technology, reduce equipment costs, reduce energy consumption and wastewater discharge, and then perform activated carbon treatment to further remove components such as pigments. Under acidic conditions, a specific cation exchange resin is used to perform ion exchange with tryptophan so that tryptophan is adsorbed on the resin. After elution with ammonia water, the obtained mixed solution is subjected to low-temperature crystallization to obtain tryptophan crystals with high purity, and then dried. After that, a tryptophan product is obtained; wherein in the ion exchange step, the cation exchange resin is a styrene-divinylphenyl resin, and then kaolin is introduced, the kaolin is pre-modified and has good dispersibility, and triethylenetetramine is introduced to enhance the compatibility with styrene monomers, so that the kaolin is better dispersed in the resin matrix, and after the sulfonation step, the resin contains more sulfonic acid groups, which can better perform ion exchange, and the prepared cation exchange resin has a large exchange capacity, high selectivity, strong stability, good compatibility with tryptophan when performing ion exchange on tryptophan, and good regeneration ability; 2. The tryptophan extracted by the method of the present invention has a purity of 99.28-99.57% and a yield of 85.1-85.6%; 3. When the cation exchange resin prepared by the method of the present invention is recycled and regenerated for the 50th time, the purity of tryptophan extracted is 94.71-96.38%, and the yield is 81.7-83.3%. DETAILED DESCRIPTION

[0014] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0015] Example 1 1. Pre-treatment Formic acid was added to the tryptophan fermentation broth to adjust the pH to 5.85, and then the temperature was reduced to 6.0°C at a rate of 2.0°C / min, and the mixture was kept warm for 14.0 hours to obtain a crystallization mixture; the crystallization mixture was centrifuged at a speed of 1550 rpm for 6.0 minutes to obtain precipitated crude crystals after the centrifugation.

[0016] 2. Decolorization The precipitated crude crystals were put into 20 times the mass of deionized water to prepare a crude crystal mixed solution, the crude crystal mixed solution was heated to 67°C, and after being fully dissolved, porous activated carbon was added and stirred at a stirring speed of 210 rpm for 20 min. After stirring, centrifugation was performed at a centrifugal speed of 4000 rpm for 10 min. After centrifugation, a decolorized solution was obtained; The porous activated carbon is 2.0% of the mass of the mixed liquid; The porous activated carbon is prepared by placing 10.0 g of activated carbon in 100 g of a 3.5 wt% hydrogen peroxide solution and stirring for 1.5 h at a temperature of 45° C. After the stirring is completed, the mixture is filtered, washed, and dried, and then kept at 120° C. for 2.0 h, and then naturally recovered to room temperature to obtain the porous activated carbon. 3. Ion exchange The decolorizing solution was adjusted to pH 3.2 with formic acid, passed into an ion exchange column, the ion exchange column was filled with a cation exchange resin, the temperature was controlled at 30°C, the flow rate was 1.6 BV / h, and after the adsorption was completed, 2.0 mol / L ammonia solution was used for elution, the elution temperature was 55°C, and the elution flow rate was 2.0 BV / h. After the elution was completed, a tryptophan mixed solution was obtained; The preparation method of the cation exchange resin includes copolymerization and sulfonation steps, which are specifically as follows: The copolymerization reaction step is to add 300g of deionized water to a reaction container, then add 0.52g of gelatin and 0.15g of sodium cocoyl methyl taurate and stir, and stir to obtain a dispersant; 27g of styrene, 9.0g of divinylbenzene, 10.8g of white oil and 0.27g of benzoyl peroxide are mixed, stirred evenly, and added to 301g of the dispersant, and the addition rate is controlled to be 1.5g / min, and then 8.2g of pre-modified kaolin is added, and homogenization treatment is performed, the homogenization time is 5min, the homogenization pressure is 1.8MPa, and the homogenization times are 2 times. After the homogenization treatment is completed, the temperature is raised to 80°C at a rate of 0.2°C / min, and the heat is kept for reaction for 7.0h. After the reaction is completed, the copolymer microspheres are cooled, washed and dried to obtain the copolymer microspheres; The preparation method of the pre-modified kaolin is as follows: calcining 20g of kaolin at 350°C for 4.0h, naturally cooling to room temperature, placing in 180g of 73wt% nitric acid solution, raising the temperature to 130°C for reflux reaction, the reaction time is 1.5h, after the reaction is completed, filtering and washing to obtain acid-washed kaolin; putting 18.0g of acid-washed kaolin into 180g of 40wt% ethanol solution, adding 1.4g of polyethylene glycol 400 and 1.1g of sodium ricinoleate, stirring evenly, adding 4.3g of triethylenetetramine, raising the temperature to 58°C, keeping warm and stirring for 1.0h, filtering, washing and drying after the insulation is completed to obtain the pre-modified kaolin; The particle size of the kaolin is 240 nm; The sulfonation step comprises placing 10.0 g of copolymer microspheres in 300 mL of ethylene dichloride, controlling the temperature to 60° C., and stirring for 2.0 h. After the insulation, adding 500 mL of 98 wt % concentrated sulfuric acid, heating to 110° C. at a rate of 1.0° C. / min, stirring to evaporate the ethylene dichloride, and insulating the reaction for 12.0 h. After the reaction is completed, cooling, filtering, washing, and drying are performed to obtain the sulfonated copolymer microspheres.

[0017] 4. Post-processing The tryptophan mixture was placed in a crystallization device for crystallization at a crystallization temperature of 2.0°C, a controlled pH of 5.95, and a crystallization time of 10.0 h. Primary tryptophan crystals were obtained by centrifugal separation. The primary tryptophan crystals were vacuum dried at a drying temperature of 80°C, a drying time of 3.2 h, and a vacuum degree of -0.08 MPa. After drying, the mixture was allowed to naturally return to room temperature to obtain a tryptophan product.

[0018] In the tryptophan fermentation broth in Example 1, the mass content of tryptophan is 54 g / L. The tryptophan extracted by the method of Example 1 has a purity of 99.57% and a yield of 85.6%.

[0019] Example 2 1. Pre-treatment Formic acid was added to the tryptophan fermentation broth to adjust the pH to 5.80, and then the temperature was reduced to 5.8°C at a rate of 1.5°C / min, and the mixture was kept warm for 13.5 hours to obtain a crystallization mixture; the crystallization mixture was centrifuged at a speed of 1500 rpm for 5.0 minutes to obtain precipitated crude crystals after the centrifugation.

[0020] 2. Decolorization The precipitated crude crystals were put into 18 times the mass of deionized water to prepare a crude crystal mixed solution, the crude crystal mixed solution was heated to 65°C, and after being fully dissolved, porous activated carbon was added and stirred at a stirring speed of 200 rpm for 18 minutes. After the stirring was completed, centrifugation was performed at a centrifugal speed of 3900 rpm for 8 minutes. After the centrifugation was completed, a decolorized solution was obtained; The porous activated carbon is 1.8% of the mass of the mixed liquid; The preparation method of the porous activated carbon is as follows: 9.5 g of activated carbon is placed in 95 g of a 3.2 wt% hydrogen peroxide solution and stirred for 1.4 h at a temperature of 43° C. After the stirring is completed, the mixture is filtered, washed, and dried, and then kept at 117° C. for 1.8 h, and then naturally recovered to room temperature to obtain the porous activated carbon; 3. Ion exchange The decolorizing solution was adjusted to pH 3.0 with formic acid, and passed into an ion exchange column filled with a cation exchange resin, and the temperature was controlled at 28°C and the flow rate was 1.5 BV / h. After the adsorption was completed, 1.8 mol / L ammonia solution was used for elution, the elution temperature was 54°C, and the elution flow rate was 1.8 BV / h. After the elution was completed, a tryptophan mixed solution was obtained; The preparation method of the cation exchange resin includes copolymerization and sulfonation steps, which are specifically as follows: The copolymerization reaction step is to add 290g of deionized water to a reaction container, then add 0.50g of gelatin and 0.14g of sodium cocoyl methyl taurate and stir, and stir to obtain a dispersant; 25g of styrene, 8.8g of divinylbenzene, 10.6g of white oil and 0.25g of benzoyl peroxide are mixed, stirred evenly, and then added to 300g of the dispersant, and the addition rate is controlled to be 1.3g / min, and then 8.0g of pre-modified kaolin is added, and homogenization treatment is performed, the homogenization time is 4min, the homogenization pressure is 1.6MPa, the homogenization times are 2 times, after the homogenization treatment is completed, the temperature is raised to 78°C at a rate of 0.1°C / min, and the heat is kept for reaction for 6.7h, and after the reaction is completed, the copolymer microspheres are cooled, washed and dried to obtain; The preparation method of the pre-modified kaolin is as follows: calcining 18g of kaolin at 345°C for 3.8h, naturally cooling to room temperature, placing in 176g of 72wt% nitric acid solution, raising the temperature to 128°C for reflux reaction, the reaction time is 1.4h, after the reaction is completed, filtering and washing to obtain acid-washed kaolin; putting 17.8g of acid-washed kaolin into 175g of 38wt% ethanol solution, adding 1.3g of polyethylene glycol 400 and 1.0g of sodium ricinoleate, stirring evenly, adding 4.1g of triethylenetetramine, raising the temperature to 56°C, keeping warm and stirring for 0.8h, filtering, washing and drying after the insulation is completed to obtain the pre-modified kaolin; The particle size of the kaolin is 234 nm; The sulfonation step comprises placing 9.8 g of copolymer microspheres in 290 mL of ethylene dichloride, controlling the temperature to 58° C., and stirring for 1.7 hours. After the heat preservation, 490 mL of 98 wt % concentrated sulfuric acid is added, and the temperature is raised to 108° C. at a rate of 0.8° C. / min. The ethylene dichloride is evaporated by stirring, and the heat preservation reaction is carried out for 11.7 hours. After the reaction is completed, the sulfonated copolymer microspheres are obtained by cooling, filtering, washing, and drying.

[0021] 4. Post-processing The tryptophan mixture was placed in a crystallization device for crystallization at a crystallization temperature of 1.8°C, a controlled pH of 5.90, and a crystallization time of 9.5 h. Primary tryptophan crystals were obtained by centrifugal separation. The primary tryptophan crystals were vacuum dried at a drying temperature of 78°C, a drying time of 3.5 h, and a vacuum degree of -0.09 MPa. After drying, the mixture was allowed to naturally return to room temperature to obtain a tryptophan product.

[0022] In the tryptophan fermentation broth in Example 2, the mass content of tryptophan is 54 g / L. The tryptophan extracted by the method of Example 2 has a purity of 99.28% and a yield of 85.1%.

[0023] Example 3 1. Pre-treatment Formic acid was added to the tryptophan fermentation broth to adjust the pH to 5.90, and then the temperature was reduced to 6.2°C at a rate of 2.5°C / min, and the mixture was kept warm for 14.5 hours to obtain a crystallization mixture; the crystallization mixture was centrifuged at a speed of 1600 rpm for 7.0 minutes, and after the centrifugation, precipitated crude crystals were obtained.

[0024] 2. Decolorization The precipitated crude crystals were put into 22 times the mass of deionized water to prepare a crude crystal mixed solution, the crude crystal mixed solution was heated to 70°C, and after being fully dissolved, porous activated carbon was added and stirred at a stirring speed of 220 rpm for 22 min. After the stirring was completed, centrifugation was performed at a centrifugal speed of 4100 rpm for 12 min. After the centrifugation was completed, a decolorized solution was obtained; The porous activated carbon is 2.2% of the mass of the mixed liquid; The preparation method of the porous activated carbon is as follows: 10.5 g of activated carbon is placed in 105 g of a 3.8 wt% hydrogen peroxide solution and stirred for 1.6 h at a temperature of 47° C. After the stirring is completed, the mixture is filtered, washed, and dried, and then kept at 123° C. for 2.2 h, and then naturally recovered to room temperature to obtain the porous activated carbon; 3. Ion exchange The decolorizing solution was adjusted to pH 3.4 with formic acid, passed into an ion exchange column, the ion exchange column was filled with a cation exchange resin, the temperature was controlled at 32°C, the flow rate was 1.7 BV / h, and after adsorption, eluted with a 2.2 mol / L ammonia solution, the elution temperature was 56°C, and the elution flow rate was 2.2 BV / h. After elution, a tryptophan mixture was obtained; The preparation method of the cation exchange resin includes copolymerization and sulfonation steps, which are specifically as follows: The copolymerization reaction step is to add 310g of deionized water to a reaction container, then add 0.55g of gelatin and 0.16g of sodium cocoyl methyl taurate and stir, and stir to obtain a dispersant; 30g of styrene, 9.2g of divinylbenzene, 11.0g of white oil and 0.30g of benzoyl peroxide are mixed, stirred evenly, and then added to 304g of the dispersant, and the addition rate is controlled to be 1.7g / min, and then 8.5g of pre-modified kaolin is added, and homogenization treatment is performed, the homogenization time is 6min, the homogenization pressure is 2.0MPa, the homogenization times are 2 times, after the homogenization treatment is completed, the temperature is raised to 82°C at a rate of 0.3°C / min, and the heat is kept for reaction for 7.3h. After the reaction is completed, the copolymer microspheres are cooled, washed and dried to obtain the copolymer microspheres; The preparation method of the pre-modified kaolin is as follows: calcining 22g of kaolin at 355°C for 4.2h, naturally cooling to room temperature, placing in 184g of 75wt% nitric acid solution, raising the temperature to 132°C for reflux reaction, the reaction time is 1.6h, after the reaction is completed, filtering and washing to obtain acid-washed kaolin; putting 18.2g of acid-washed kaolin into 185g of 42wt% ethanol solution, adding 1.5g of polyethylene glycol 400 and 1.2g of sodium ricinoleate, stirring evenly, adding 4.5g of triethylenetetramine, raising the temperature to 60°C, keeping warm and stirring for 1.2h, filtering, washing and drying after the insulation is completed to obtain the pre-modified kaolin; The particle size of the kaolin is 245 nm; The sulfonation step comprises placing 10.3 g of copolymer microspheres in 310 mL of ethylene dichloride, controlling the temperature to 62° C., and stirring for 2.3 hours. After the heat preservation, 510 mL of 98 wt % concentrated sulfuric acid is added, and the temperature is raised to 113° C. at a rate of 1.2° C. / min. The ethylene dichloride is evaporated by stirring, and the heat preservation reaction is carried out for 12.3 hours. After the reaction is completed, the sulfonated copolymer microspheres are obtained by cooling, filtering, washing, and drying.

[0025] 4. Post-processing The tryptophan mixture was placed in a crystallization device for crystallization at a crystallization temperature of 2.2°C, a controlled pH of 6.0, and a crystallization time of 10.5 h. Primary tryptophan crystals were obtained by centrifugation. The primary tryptophan crystals were vacuum dried at a drying temperature of 82°C, a drying time of 3.0 h, and a vacuum degree of -0.06 MPa. After drying, the mixture was allowed to naturally return to room temperature to obtain a tryptophan product.

[0026] In the tryptophan fermentation broth in Example 3, the mass content of tryptophan is 54 g / L. The tryptophan extracted by the method of Example 3 has a purity of 99.46% and a yield of 85.3%.

[0027] Comparative Example 1 Based on Example 1, the changes are as follows: In the ion exchange step, the cation exchange resin was replaced with 001×7 strong acid cation exchange resin, and the rest of the operations were the same.

[0028] In the tryptophan fermentation broth in Comparative Example 1, the mass content of tryptophan is 54 g / L. The tryptophan extracted by the method of Comparative Example 1 has a purity of 90.14% and a yield of 72.2%.

[0029] Comparative Example 2 Based on Example 1, the change is that in the preparation method of the cation exchange resin in the ion exchange step, In the copolymerization step, gelatin and sodium cocoyl methyl taurate in the dispersant were replaced with deionized water in equal amounts; An equal amount of premodified kaolin was replaced by acid-washed kaolin; The rest of the operations are the same.

[0030] In the tryptophan fermentation broth in Comparative Example 2, the mass content of tryptophan was 54 g / L. The tryptophan extracted by the method of Comparative Example 2 had a purity of 92.48% and a yield of 76.4%.

[0031] Cation exchange resin regeneration performance test The macroporous exchange resins prepared in Examples 1-3 and Comparative Examples 1-2 were recycled and regenerated for 50 times, and the macroporous exchange resins were recycled and regenerated after each ion exchange step; The specific regeneration method is to place the cation exchange resin after ion exchange in 4 times the volume of 5wt% sodium hydroxide solution and stir for 4.0 hours, filter and wash after the stirring, and then place it in 4 times the volume of 5wt% hydrochloric acid solution and stir for 4.0 hours, filter and wash after the stirring, and dry at 110°C to obtain the regenerated resin; At the 50th recycling, the yield and purity of tryptophan obtained were as follows:

[0032] The invention directly performs low-temperature crystallization on tryptophan fermentation liquid, and through centrifugation, most of the components such as bacteria, proteins, pigments, etc. in the fermentation liquid are left in the mother liquor, so that separation from tryptophan is achieved, filtration by ultrafiltration membrane technology is avoided, the cost of equipment is reduced, energy consumption and wastewater discharge are reduced, and then the pigments and other components are further removed through activated carbon treatment. Under acidic conditions, a specific cation exchange resin is used to perform ion exchange with tryptophan so that tryptophan is adsorbed on the resin. After elution with ammonia water, the obtained mixed liquid is subjected to low-temperature crystallization to obtain tryptophan crystals with higher purity. After drying, , and obtain a tryptophan product; wherein in the ion exchange step, the cation exchange resin is a styrene-divinylphenyl resin, and then kaolin is introduced, the kaolin is pre-modified and has good dispersibility, and triethylenetetramine is introduced to enhance the compatibility with styrene monomers, etc., so that the kaolin is better dispersed in the resin matrix, and after the sulfonation step, the resin contains more sulfonic acid groups, which can better perform ion exchange, and the prepared cation exchange resin has a large exchange capacity, high selectivity, strong stability, good compatibility with tryptophan when performing ion exchange on tryptophan, and good regeneration ability.

[0033] Comparative Example 1 directly uses 001×7 strong acid ion exchange resin for ion exchange. Compared with the specific ion exchange resin of the present invention, it has poor exchange capacity and regeneration ability, and the adsorption selectivity for tryptophan is not high, thereby reducing the yield and purity of tryptophan; Comparative Example 2 only uses acid-washed kaolin and styrene-divinylbenzene for cross-linking, and its dispersibility in the resin matrix is ​​poor, which affects the completeness and uniformity of the copolymerization and cross-linking, and affects the stability, adsorption and stability of the cation exchange resin, and ultimately affects the purity and yield of the tryptophan product and reduces the regeneration ability.

[0034] Unless otherwise specified, all ratios and percentages described in the present invention are by mass ratios and percentages are by mass percentages.

[0035] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for separating and extracting tryptophan from a fermentation broth, characterized in that: The method comprises pre-treatment, decolorization, ion exchange and post-treatment steps; The pretreatment step comprises adding formic acid to the tryptophan fermentation broth, adjusting the pH to 5.80-5.90, then lowering the temperature to 5.8-6.2° C., keeping the temperature and standing for 13.5-14.5 hours to obtain a crystallization mixture; centrifuging the crystallization mixture to obtain precipitated crude crystals; The decolorization step comprises: putting the precipitated crude crystals into deionized water, fully dissolving them, adding porous activated carbon, stirring, and centrifuging to obtain a decolorized solution; The ion exchange step comprises: adjusting the pH of the decolorizing solution to 3.0-3.4 with formic acid, passing the solution into an ion exchange column, filling the ion exchange column with a cation exchange resin, controlling the temperature to 28-32° C., and the flow rate to 1.5-1.7 BV / h; and eluting with a 1.8-2.2 mol / L ammonia solution after adsorption, at a temperature of 54-56° C. and an elution flow rate of 1.8-2.2 BV / h. After elution, a tryptophan mixture is obtained; The preparation method of the cation exchange resin comprises copolymerization and sulfonation steps; The copolymerization reaction steps are as follows: adding deionized water to a reaction container, then adding gelatin and sodium cocoyl methyl taurate and stirring, stirring evenly to obtain a dispersant; mixing styrene, divinylbenzene, white oil and benzoyl peroxide, stirring evenly, adding the mixture to the dispersant, controlling the adding rate to be 1.3-1.7 g / min, then adding pre-modified kaolin, and homogenizing, and after the homogenization is completed, heating to 78-82° C. at a rate of 0.1-0.3° C. / min, and keeping the temperature for reaction for 6.7-7.3 hours, and after the reaction is completed, cooling, washing and drying to obtain copolymer microspheres; The preparation method of the pre-modified kaolin comprises the following steps: calcining the kaolin, and then placing it in a nitric acid solution for reflux reaction, filtering and washing after the reaction is completed to obtain acid-washed kaolin; placing the acid-washed kaolin in an ethanol solution, adding polyethylene glycol 400 and sodium ricinoleate, stirring evenly, adding triethylenetetramine, raising the temperature to 56-60° C., keeping the temperature and stirring for 0.8-1.2 hours, filtering, washing and drying after the heat preservation is completed to obtain the pre-modified kaolin; The post-treatment step is to crystallize and dry the tryptophan mixture to obtain a tryptophan product.

2. A method for separating and extracting tryptophan from a fermentation broth according to claim 1, characterized in that: The pretreatment step comprises adding formic acid to the tryptophan fermentation broth, adjusting the pH to 5.80-5.90, then reducing the temperature to 5.8-6.2°C at a rate of 1.5-2.5°C / min, keeping the temperature and standing for 13.5-14.5 hours to obtain a crystallization mixture; and centrifuging the crystallization mixture at a speed of 1500-1600 rpm for 5.0-7.0 minutes to obtain precipitated crude crystals after the centrifugation.

3. A method for separating and extracting tryptophan from a fermentation broth according to claim 1, characterized in that: The decolorization step comprises: placing the precipitated crude crystals into 18-22 times the mass of deionized water to obtain a crude crystal mixed solution, raising the temperature of the crude crystal mixed solution to 65-70° C., adding porous activated carbon after the crude crystal mixed solution is fully dissolved, stirring at a stirring speed of 200-220 rpm for 18-22 min, centrifuging after the stirring is completed at a centrifugal speed of 3900-4100 rpm for 8-12 min, and obtaining a decolorized solution after the centrifugation is completed; The porous activated carbon accounts for 1.8-2.2% of the mass of the mixed liquid.

4. A method for separating and extracting tryptophan from a fermentation broth according to claim 1, characterized in that: The preparation method of the porous activated carbon is as follows: placing the activated carbon in a hydrogen peroxide solution and stirring it for 1.4-1.6 hours at a stirring temperature of 43-47° C. After the stirring is completed, filtering, washing, and drying, the mixture is kept at 117-123° C. for 1.8-2.2 hours, and then naturally recovered to room temperature to obtain the porous activated carbon; The mass ratio of the activated carbon to the hydrogen peroxide solution is 9.5-10.5:95-105; The mass concentration of the hydrogen peroxide solution is 3.2-3.8%.

5. The method for separating and extracting tryptophan from a fermentation broth according to claim 1, characterized in that: In the preparation method of the cation exchange resin, in the copolymerization step, the homogenization treatment has a homogenization time of 4-6 minutes, a homogenization pressure of 1.6-2.0 MPa, and a homogenization frequency of 2 times.

6. The method for separating and extracting tryptophan from a fermentation broth according to claim 1, characterized in that: In the preparation method of the cation exchange resin, the mass ratio of the deionized water, gelatin, and sodium cocoyl methyl taurate is 290-310:0.50-0.55:0.14-0.16; The mass ratio of the styrene, divinylbenzene, white oil, benzoyl peroxide, dispersant and pre-modified kaolin is 25-30:8.8-9.2:10.6-11.0:0.25-0.30:300-304:8.0-8.

5.

7. The method for separating and extracting tryptophan from a fermentation broth according to claim 1, characterized in that: In the preparation method of the pre-modified kaolin, the calcination temperature is 345-355° C. and the calcination time is 3.8-4.2 h; The reflux reaction has a reaction temperature of 128-132° C. and a reaction time of 1.4-1.6 h.

8. The method for separating and extracting tryptophan from a fermentation broth according to claim 1, characterized in that: In the preparation method of the pre-modified kaolin, the particle size of the kaolin is 234-245 nm; The mass ratio of kaolin to nitric acid solution is 18-22:176-184; The mass concentration of the nitric acid solution is 72-75%; The mass ratio of the acid-washed kaolin, ethanol solution, polyethylene glycol 400, sodium castor oil sulfonate and triethylenetetramine is 17.8-18.2:175-185:1.3-1.5:1.0-1.2:4.1-4.5; The mass concentration of the ethanol solution is 38-42%.

9. The method for separating and extracting tryptophan from a fermentation broth according to claim 1, characterized in that: The sulfonation step comprises placing the copolymer microspheres in ethylene dichloride, controlling the temperature to 58-62° C., keeping the temperature and stirring for 1.7-2.3 hours, adding concentrated sulfuric acid after the temperature is kept at 108-113° C. at a rate of 0.8-1.2° C. / min, stirring to evaporate the ethylene dichloride, keeping the temperature and reacting for 11.7-12.3 hours, and cooling, filtering, washing and drying after the reaction to obtain the sulfonated copolymer microspheres; The mass volume ratio of the copolymer microspheres, ethylene dichloride and concentrated sulfuric acid is 9.8-10.3 g: 290-310 mL: 490-510 mL; The mass concentration of the concentrated sulfuric acid is 98%.

10. The method for separating and extracting tryptophan from a fermentation broth according to claim 1, characterized in that: The post-treatment step is to place the tryptophan mixture in a crystallization device for crystallization, the crystallization temperature is 1.8-2.2° C., the pH is controlled to be 5.90-6.0, the crystallization time is 9.5-10.5 hours, primary tryptophan crystals are obtained by centrifugal separation, the primary tryptophan crystals are vacuum dried, the drying temperature is 78-82° C., the drying time is 3.0-3.5 hours, the vacuum degree is -0.06 to -0.09 MPa, and after drying, the tryptophan product is obtained after naturally returning to room temperature.

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