Purification method of high-purity tryptophan

Abstract

The invention relates to a purification method of high-purity tryptophan, and relates to the technical field of tryptophan purification, and the purification method comprises the following steps: S1, microfiltration; s2, performing ultrafiltration; s3, adsorbing tryptophan by using ion exchange resin; s4, elution is carried out; s5, decoloring; s6, crystallizing; s7, drying; the ion exchange resin is strong acid dodecyl styrene-divinyl benzene resin. The purification effect of the ion exchange resin on the tryptophan is improved, so that the high-purity tryptophan is prepared.

Description

Technical Field

[0001] The present application relates to the field of tryptophan purification technology, and in particular to a method for purifying high-purity tryptophan. Background Art

[0002] Tryptophan, also known as α-amino-β-indolepropionic acid, has a molecular weight of 204.23. L-Tryptophan is a white or slightly yellow leaf-shaped crystal or powder, odorless or slightly odorous. It is soluble in boiling water and hot ethanol, and slightly soluble in water. L-Tryptophan is one of the essential amino acids in the life activities of humans and animals. It plays an important role in the growth and development, growth and metabolism of humans and animals, and is widely used in medicine, food and feed. At present, the production methods of L-Tryptophan include chemical synthesis, protein decomposition, microbial transformation, enzyme method and direct fermentation method, among which direct fermentation method is the mainstream method of tryptophan production.

[0003] At present, the method for purifying L-tryptophan in fermentation broth mainly adopts a series of processes such as filtration, ion exchange resin adsorption, elution, decolorization, crystallization and drying. Among them, ion exchange resin has the problem of poor separation of inorganic salts and other miscellaneous acids (glutamic acid), thereby reducing the purity of tryptophan. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a method for purifying high-purity tryptophan.

[0005] The present application provides a method for purifying high-purity tryptophan, which adopts the following technical scheme: A method for purifying high-purity tryptophan, the method comprising the following steps: S1. Microfiltration: The tryptophan fermentation broth is adjusted to pH 3.0-3.5 with acid, filtered using a microfiltration membrane, and the microfiltration clear liquid is collected; S2. Ultrafiltration: ultrafiltration the collected microfiltration clear liquid using an ultrafiltration membrane to collect the ultrafiltration clear liquid; S3. Ion exchange resin: use ion exchange resin to adsorb tryptophan; S4. Elution: eluting with aqueous ammonia at a concentration of 2.0-2.5 mol / L, collecting the crude tryptophan extract; S5. Decolorization: decolorizing the crude tryptophan extract with activated carbon, and collecting the decolorized clear solution; S6. Crystallization: The decolorized clear solution is concentrated and crystallized under vacuum; S7. Drying: After drying, high-purity tryptophan is obtained; The ion exchange resin is a strongly acidic dodecylstyrene-divinylbenzene resin.

[0006] By adopting the above technical scheme, the present application found in the experimental process that the purity of tryptophan purified by using 001×7 cation exchange resin was reduced by 2.5% compared with that by using strong acid dodecylstyrene-divinylbenzene resin, and the purity of tryptophan purified by using D061 cation exchange resin was reduced by 2.2% compared with that by using strong acid dodecylstyrene-divinylbenzene resin. It may be that the alkyl group of the strong acid dodecylstyrene-divinylbenzene resin in Example 1.3 improves the π-π interaction and hydrophobic effect between the benzene ring of the polymer and the indole ring in tryptophan, thereby making the selective adsorption effect of the strong acid dodecylstyrene-divinylbenzene resin on tryptophan better, improving the separation effect of tryptophan from inorganic salts and miscellaneous acids, and thus improving the purity of tryptophan.

[0007] Preferably, the preparation method of the strongly acidic dodecylstyrene-divinylbenzene resin comprises the following steps: A1. Stir and mix water and gelatin evenly, react for 13-17 minutes, then add cellulose to completely dissolve it to obtain a stabilizer solution; A2. The mixture of dodecylstyrene and divinylbenzene was mixed, rinsed 2-3 times with a 4-6% sodium hydroxide solution, and then rinsed 2-3 times with water; A3. The mixture and the initiator are added to the stabilizer solution, the stabilizer solution is used as the reaction solvent, and the amount thereof is such that the mixture and the initiator can be evenly dispersed, and the mixture is heated to 60-65°C at a rate of 80-90r / min with stirring, and kept warm for 2.0-2.5h, and then heated to 70-75°C, and kept warm for 6.0-6.5h, and then heated to 85-90°C, and kept warm for 1.0-1.5h, and then filtered after cooling, rinsed and dried to obtain macroporous cross-linked styrene white balls; A4. The macroporous cross-linked styrene white ball is swollen with a swelling agent, and concentrated sulfuric acid at a concentration of 95-98% is added under stirring, and the reaction temperature is 80-90 ° C. After 10-12h, the temperature is lowered, and then the concentrated sulfuric acid is diluted to obtain a strongly acidic dodecylstyrene-divinylbenzene resin; Wherein, in step A1, the mass ratio of gelatin, cellulose and water is (8-10):(0.8-1.2):(135-145); in step A2, the mass of divinylbenzene is 0.8-0.9% of the mass of dodecylstyrene; and in step A3, the mass of the initiator is 0.8%-1% of the mass of the mixture.

[0008] By adopting the above technical solution, the present application found in the experimental process that when the mass ratio of gelatin, cellulose and water in step A1 is lower than the range of (8-10):(0.8-1.2):(135-145) provided in the present application, the purity of tryptophan purified by the prepared strongly acidic dodecylstyrene-divinylbenzene resin is reduced by 0.7% and 0.8% compared with the mass ratio of gelatin, cellulose and water in step A1 of 8:1.2:135 and 10:0.8:145, respectively. When the mass ratio of gelatin, cellulose and water in step A1 is higher than the range of (8-10):(0.8-1.2):(135-145) provided in the present application, the purity of tryptophan purified by the prepared strongly acidic dodecylstyrene-divinylbenzene resin is reduced by 0.7% and 0.8% compared with the mass ratio of gelatin, cellulose and water in step A1 of 8:1.2:135 and 10:0.8:145, respectively. 2):(135-145), the purity of tryptophan purified by the obtained strong acid dodecylstyrene-divinylbenzene resin was reduced by 0.8% and 0.9% respectively compared with the case where the mass ratio of gelatin, cellulose and water in step A1 was 8:1.2:135 and 10:0.8:145. This may be because when the mass ratio of gelatin, cellulose and water in step A1 was (8-10):(0.8-1.2):(135-145), the particle size distribution of the dodecylstyrene-divinylbenzene polymer was more uniform, thereby improving the purification effect of the strong acid dodecylstyrene-divinylbenzene resin on tryptophan.

[0009] The present application found during the experiment that when the mass of divinylbenzene in step A2 is lower than 0.8% of the mass of dodecylstyrene, the purity of tryptophan purified from the obtained strong acid dodecylstyrene-divinylbenzene resin is reduced by 1.2% and 1.3% respectively compared with the case where the mass of divinylbenzene is 0.8% and 0.9% of the mass of dodecylstyrene, and when the mass of divinylbenzene in step A2 is higher than 0.9% of the mass of dodecylstyrene, the purity of tryptophan purified from the obtained strong acid dodecylstyrene-divinylbenzene resin is reduced by 1.1% and 1.2% respectively compared with the case where the mass of divinylbenzene is 0.8% and 0.9% of the mass of dodecylstyrene, which may be because when the mass of divinylbenzene in step A2 is 0.8-0.9% of the mass of dodecylstyrene, the crosslinking degree of the dodecylstyrene-divinylbenzene polymer is optimized, thereby improving the purification effect of the strong acid dodecylstyrene-divinylbenzene resin on tryptophan.

[0010] During the experiment, the present application found that when the mass of the initiator in step A3 is less than 0.8% of the mass of the mixture, the purity of the tryptophan obtained by the purified strong acid dodecylstyrene-divinylbenzene resin is reduced by 0.9% and 1.1% respectively compared with the case where the mass of the initiator in step A3 is 0.8% and 1% of the mass of the mixture, and when the mass of the initiator in step A3 is higher than 1% of the mass of the mixture, the purity of the purified tryptophan is reduced by 0.8% and 1% respectively compared with the case where the mass of the initiator in step A3 is 0.8% and 1% of the mass of the mixture, which may be because when the mass of the initiator in step A3 is 0.8%-1% of the mass of the mixture, the pore structure of the strongly acid dodecylstyrene-divinylbenzene resin is optimized, thereby improving the purification effect of tryptophan.

[0011] The present application found during the experiment that when the stirring speed in step A3 is lower than 80 r / min, the purity of tryptophan purified from the obtained strong acid dodecyl styrene-divinyl benzene resin is reduced by 0.8% and 0.9% respectively compared with the stirring speed at 80 r / min and 90 r / min in step A3. When the stirring speed in step A3 is higher than 90 r / min, the purity of tryptophan purified from the obtained strong acid dodecyl styrene-divinyl benzene resin is reduced by 0.7% and 0.8% respectively compared with the stirring speed at 80 r / min and 90 r / min in step A3. It may be that when stirring at a speed of 80-90 r / min in step A3, the particle size range of the strong acid dodecyl styrene-divinyl benzene resin is optimized, thereby improving the purification effect of tryptophan.

[0012] During the experiment, the present application found that when the reaction temperature of step A4 is lower than 80°C, the purity of tryptophan purified from the obtained strong acid dodecylstyrene-divinylbenzene resin is reduced by 0.7% and 0.8% respectively compared with the case when the reaction temperature of step A4 is 80-90°C. When the reaction temperature of step A4 is higher than 90°C, the purity of tryptophan purified from the obtained strong acid dodecylstyrene-divinylbenzene resin is reduced by 0.9% and 1% respectively compared with the case when the reaction temperature of step A4 is 80-90°C. It may be that when the reaction temperature of step A4 is 80-90°C, the degree of sulfonation of the strong acid dodecylstyrene-divinylbenzene resin is optimized, thereby improving the purification effect of tryptophan.

[0013] Preferably, in step A1, the mass ratio of gelatin, cellulose and water is 9:1:140.

[0014] By adopting the above technical scheme, the present application found in the experimental process that when the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the purity of tryptophan purified by the obtained strong acid dodecylstyrene-divinylbenzene resin is 0.4% and 0.3% higher than that when the mass ratio of gelatin, cellulose and water in step A1 is 8:1.2:135 and 10:0.8:145, respectively. It may be that when the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the particle size distribution of the strong acid dodecylstyrene-divinylbenzene resin is further optimized, thereby further improving the purification effect of tryptophan.

[0015] Preferably, in step A2, the mass of divinylbenzene is 0.85% of the mass of dodecylstyrene.

[0016] By adopting the above technical scheme, the present application found in the experimental process that when the mass of divinylbenzene is 0.85% of the mass of dodecylstyrene, the purity of tryptophan purified by the prepared strong acid dodecylstyrene-divinylbenzene resin is increased by 0.3% and 0.2% respectively compared with when the mass of divinylbenzene is 0.8% and 0.9% of the mass of dodecylstyrene. It may be that the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, and the mass of divinylbenzene is 0.85% of the mass of dodecylstyrene, which further optimizes the purification effect of strong acid dodecylstyrene-divinylbenzene resin on tryptophan.

[0017] Preferably, the mass of the initiator is 0.9% of the mass of the mixture.

[0018] By adopting the above technical scheme, the present application found in the experimental process that when the mass of the initiator in step A3 is 0.9% of the mass of the mixture, the purity of tryptophan purified by the prepared strong acid dodecylstyrene-divinylbenzene resin is increased by 0.4% and 0.2% respectively compared with when the mass of the initiator is 0.8% and 1% of the mass of the mixture. It may be that the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the mass of divinylbenzene in step A2 is 0.85% of the mass of dodecylstyrene, and the mass of the initiator in step A3 is 0.9% of the mass of the mixture, which further optimizes the purification effect of the strong acid dodecylstyrene-divinylbenzene resin on tryptophan.

[0019] Preferably, the stirring speed in step A3 is 85 r / min.

[0020] By adopting the above technical scheme, the present application found in the experimental process that when stirring at a speed of 85 r / min in step A3, the purity of tryptophan purified by the obtained strong acid dodecylstyrene-divinylbenzene resin was increased by 0.4% and 0.3% respectively compared with stirring at a speed of 80 r / min and 90 r / min in step A3. This may be because the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the mass of divinylbenzene in step A2 is 0.85% of the mass of dodecylstyrene, the mass of the initiator in step A3 is 0.9% of the mass of the mixture, and when stirring at a speed of 85 r / min in step A3, the purification effect of the strong acid dodecylstyrene-divinylbenzene resin on tryptophan is further optimized.

[0021] Preferably, the reaction temperature in step A4 is 85°C.

[0022] By adopting the above technical scheme, the present application found in the experimental process that when the reaction temperature in step A4 is 85°C, the purity of tryptophan purified by the obtained strong acid dodecylstyrene-divinylbenzene resin is increased by 0.3% and 0.2% respectively compared with when the reaction temperature in step A4 is 80°C and 90°C. This may be because the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the mass of divinylbenzene in step A2 is 0.85% of the mass of dodecylstyrene, the mass of the initiator in step A3 is 0.9% of the mass of the mixture, and the stirring speed in step A3 is 85r / min. When the reaction temperature in step A4 is 85°C, the purification effect of the strong acid dodecylstyrene-divinylbenzene resin on tryptophan is further optimized.

[0023] Preferably, in step S5, the amount of activated carbon added is 1.2-1.4%, the decolorization temperature is 55-60° C., and the decolorization time is 45-50 min.

[0024] Preferably, the concentration at the end point of concentration in step S6 is 50-55 g / L.

[0025] In summary, this application has the following beneficial technical effects: The present application prepares a strongly acidic dodecylstyrene-divinylbenzene resin for purifying tryptophan. When the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the mass of divinylbenzene in step A2 is 0.85% of the mass of dodecylstyrene, the mass of the initiator in step A3 is 0.9% of the mass of the mixture, stirring is performed at a speed of 85 r / min in step A3, and the reaction temperature in step A4 is 85° C., the prepared strongly acidic dodecylstyrene-divinylbenzene resin has an excellent effect on purifying tryptophan, thereby improving the purity of tryptophan. DETAILED DESCRIPTION

[0026] The specific embodiments are merely explanations of the present application and are not limitations of the present application. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, as long as they are within the scope of the claims of the present application, they are protected by the patent law.

[0027] Material Source PVDF 0.2 μm microfiltration membrane; ultrafiltration membrane, product model PLCGC10205; cellulose is methyl hydroxyethyl cellulose, CAS number 9032-42-2 and divinylbenzene were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Ammonia was purchased from Shandong Hengshuo Chemical Co., Ltd.; Activated carbon, with a specification of 200 mesh powdered activated carbon, was purchased from Kelin Water Purification Materials Co., Ltd.; Gelatin, industrial grade, model 9000-7-8, purchased from Shandong Poly Chemical Co., Ltd.; In the preparation example of this application, dibenzoyl peroxide was selected as the initiator, and dibenzoyl peroxide was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; The preparation of dodecyl styrene is based on Pan Leiming, Ma Chao, Zhou Chao, et al. Synthesis and structural characterization of dodecyl styrene with hydrophobic monomers [J]. Fine Petrochemicals, 2018, 35(02): 33-35. The method for preparing dodecylstyrene comprises the following steps: 1. Synthesis of p-Dodecylacetophenone 40.002g of anhydrous aluminum chloride was added to a three-necked round-bottom flask containing 200mL of dichloromethane under a constant temperature of 0°C. After stirring for 30min, a mixed solution of dichloromethane and acetic anhydride (25mL of dichloromethane, 20.418g of acetic anhydride) was slowly added dropwise. After the addition was completed and stirred for 15min, a mixed solution of dichloromethane and dodecylbenzene (25mL of dichloromethane, 24.643g of dodecylbenzene) was slowly added dropwise. After the addition was completed, the mixture was heated to 30°C for reaction for 5h. After the reaction was completed, the system was cooled to 0°C, and then filtered to remove solid impurities, and washed twice with 150mL of 10% hydrochloric acid solution to remove anhydrous aluminum chloride and acetic anhydride, washed twice with 150mL of saturated sodium carbonate solution to remove residual hydrochloric acid, and washed twice with saturated sodium chloride solution to remove excess impurities (separating funnel was used to separate and remove the lower layer of solution after each washing). The resulting solution was dehydrated with anhydrous magnesium sulfate and filtered to remove anhydrous magnesium sulfate and other impurities. Finally, the dichloromethane solvent was removed by vacuum rotary evaporation to obtain transparent light red p-dodecylacetophenone.

[0028] 1. Synthesis of 1-methyl-p-dodecylbenzyl alcohol 14.418g of p-dodecylacetophenone was added to a three-necked round-bottom flask containing 250mL of methanol at a constant temperature of 0℃, stirred and 5 portions of 1.135gg of sodium borohydride were added in sequence. The temperature was raised to 60℃ and the reaction was carried out at a constant temperature for 2h. After the reaction was completed, the initial product was vacuum-evaporated to remove the methanol solvent to obtain a yellow block solid. The solid was dissolved in 250mL of n-hexane and washed twice with 150mL of hydrochloric acid solution to remove sodium borohydride, washed twice with 150mL of saturated sodium carbonate solution to remove residual hydrochloric acid, and washed twice with saturated sodium chloride solution to remove excess impurities (separate the upper solution with a separatory funnel after each washing). The solution obtained after washing was dehydrated with anhydrous magnesium sulfate and then filtered to remove anhydrous magnesium sulfate and other impurities. Finally, the n-hexane solvent was vacuum-evaporated to obtain transparent light-dark yellow 1-methyl-p-dodecylbenzyl alcohol.

[0029] 3. Synthesis of n-dodecylstyrene Add 7.259g of 1-methyl-p-dodecylbenzyl alcohol to a single-mouth round-bottom flask containing 350mL of xylene, place the flask in a heat-collecting constant temperature heating magnetic stirrer, add 9.511g of p-toluenesulfonic acid monohydrate to the flask, and condense and reflux for 2h at a constant temperature of 160°C. After the reaction is completed, cool to room temperature, wash twice with 150mL of distilled water, separate and take the upper solution. The obtained solution is dehydrated with anhydrous magnesium sulfate and then filtered to remove anhydrous magnesium sulfate and other impurities. Finally, vacuum rotary evaporation is used to remove the xylene solvent to obtain a black solution. After separation and purification by silica gel column chromatography with n-hexane as the chromatographic liquid, p-dodecylstyrene is obtained: a colorless, transparent, oily liquid with an aromatic odor, flammable, insoluble in water, and soluble in organic solvents such as toluene.

[0030] Preparation Example 1.1 Strongly acidic dodecylstyrene-divinylbenzene resin was prepared by the following method: A1. 1404.3 mL of water and 83.22 g of gelatin were stirred and mixed evenly. After reacting for 15 min, 12.48 g of cellulose was added to completely dissolve it to obtain a stabilizer solution; A2. 491.4 g of dodecylstyrene and 4.18 g of divinylbenzene were mixed to obtain a mixture, which was rinsed three times with a 5% sodium hydroxide solution and then rinsed three times with water; A3. The mixture and 4.42 g of dibenzoyl peroxide were added to the stabilizer solution, stirred at a rate of 85 r / min and heated to 60 ° C, kept warm for 2.5 h, then heated to 75 ° C, kept warm for 6.0 h, then heated to 85 ° C, kept warm for 1.5 h, cooled and filtered, rinsed and dried to obtain macroporous cross-linked styrene white balls; A4. Use 1,2-dichloroethane to swell the macroporous cross-linked styrene white ball, add 98% concentrated sulfuric acid under stirring, react at a reaction temperature of 85°C for 12 hours, then cool down, and then dilute the concentrated sulfuric acid to obtain a strongly acidic dodecylstyrene-divinylbenzene resin.

[0031] Preparation Example 1.2 The difference from Preparation Example 1.1 is that in step A1, the water is 1396 mL, the gelatin is 96.3 g, and the cellulose is 7.7 g.

[0032] Preparation Example 1.3 The difference from Preparation Example 1.1 is that in step A1, the water is 1400 mL, the gelatin is 90 g, and the cellulose is 10 g.

[0033] Preparation Example 1.4 The difference from Preparation Example 1.1 is that in step A1, the water is 1406.3 mL, the gelatin is 70.31 g, and the cellulose is 23.44 g.

[0034] Preparation Example 1.5 The difference from Preparation Example 1.1 is that in step A1, the amount of water is 1384.6 mL, the amount of gelatin is 110.77 g, and the amount of cellulose is 4.63 g.

[0035] Preparation Example 2.1 The difference from Preparation Example 1.3 is that the amount of divinylbenzene in Step A2 is 3.93 g.

[0036] Preparation Example 2.2 The difference from Preparation Example 1.3 is that the amount of divinylbenzene in Step A2 is 4.42 g.

[0037] Preparation Example 2.3 The difference from Preparation Example 1.3 is that the amount of divinylbenzene in Step A2 is 2.95 g.

[0038] Preparation Example 2.4 The difference from Preparation Example 1.3 is that the amount of divinylbenzene in Step A2 is 5.41 g.

[0039] Preparation Example 3.1 The difference from Preparation Example 1.3 is that in Step A3, the amount of dibenzoyl peroxide is 3.93 g.

[0040] Preparation Example 3.2 The difference from Preparation Example 1.3 is that the amount of dibenzoyl peroxide in Step A3 is 4.91 g.

[0041] Preparation Example 3.3 The difference from Preparation Example 1.3 is that the amount of dibenzoyl peroxide in Step A3 is 2.95 g.

[0042] Preparation Example 3.4 The difference from Preparation Example 1.3 is that the amount of dibenzoyl peroxide in Step A3 is 5.9 g.

[0043] Preparation Example 4.1 The difference from Preparation Example 1.3 is that in step A3, the stirring speed is 80 r / min.

[0044] Preparation Example 4.2 The difference from Preparation Example 1.3 is that in step A3, the stirring speed is 90 r / min.

[0045] Preparation Example 4.3 The difference from Preparation Example 1.3 is that in step A3, the stirring speed is 70 r / min.

[0046] Preparation Example 4.4 The difference from Preparation Example 1.3 is that in step A3, the stirring speed is 100 r / min.

[0047] Preparation Example 5.1 The difference from Preparation Example 1.3 is that the reaction temperature in Step A4 is 80°C.

[0048] Preparation Example 5.2 The difference from Preparation Example 1.3 is that the reaction temperature in Step A4 is 90°C.

[0049] Preparation Example 5.3 The difference from Preparation Example 1.3 is that the reaction temperature in Step A4 is 70°C.

[0050] Preparation Example 5.4 The difference from Preparation Example 1.3 is that the reaction temperature in Step A4 is 100°C.

[0051] Example 1.1 The method for purifying high-purity tryptophan comprises the following steps: S1. Microfiltration: The tryptophan fermentation broth was adjusted to pH 3.0 with acid, filtered using a PVDF 0.2 μm microfiltration membrane at 25°C and an operating pressure of 0.1 MPa, and the microfiltration clear liquid was collected; S2. Ultrafiltration: ultrafiltration of the collected microfiltration clear liquid using an ultrafiltration membrane at 25°C and an operating pressure of 0.1 MPa, and collecting the ultrafiltration clear liquid; S3. Ion exchange resin: use ion exchange resin to adsorb tryptophan; S4. Elution: eluting with 2.0 mol / L ammonia water to collect the crude tryptophan extract; S5. Decolorization: adding activated carbon to the crude tryptophan extract at 55°C to decolorize the crude tryptophan extract for 45 minutes, and collecting the decolorized clear liquid; S6. Crystallization: The decolorized supernatant was concentrated and crystallized under vacuum, and the concentration at the end point was 50 g / L to obtain crystals and recover the mother liquor; S7. Drying: drying the crystals obtained in step S6 by a dryer to obtain high-purity tryptophan; The ion exchange resin in step S3 is the strongly acidic dodecylstyrene-divinylbenzene resin prepared in Preparation Example 1.1.

[0052] Examples 1.2-1.5 The difference from Example 1.3 is that the ion exchange resins used in Examples 1.2-1.5 are the strongly acidic dodecylstyrene-divinylbenzene resins prepared in Preparation Examples 1.2-1.5 respectively.

[0053] Examples 2.1-2.4 The difference from Example 1.3 is that the ion exchange resins used in Examples 2.1-2.4 are the strongly acidic dodecylstyrene-divinylbenzene resins prepared in Preparation Examples 2.1-2.4, respectively.

[0054] Examples 3.1-3.4 The difference from Example 1.3 is that the ion exchange resins used in Examples 3.1-3.4 are the strongly acidic dodecylstyrene-divinylbenzene resins prepared in Preparation Examples 3.1-3.4, respectively.

[0055] Examples 4.1-4.4 The difference from Example 1.3 is that the ion exchange resins used in Examples 4.1-4.4 are the strongly acidic dodecylstyrene-divinylbenzene resins prepared in Preparation Examples 4.1-4.4, respectively.

[0056] Examples 5.1-5.4 The difference from Example 1.3 is that the ion exchange resins used in Examples 5.1-5.4 are the strongly acidic dodecylstyrene-divinylbenzene resins prepared in Preparation Examples 5.1-5.4, respectively.

[0057] Comparative Example 1-2 The difference from Example 1.3 is that the ion exchange resins used in Comparative Example 1-2 are 001×7 cation exchange resin and D061 cation exchange resin, respectively.

[0058] Performance Testing The purity of tryptophan obtained in Examples 1.1-1.5, Examples 2.1-2.4, Examples 3.1-3.4, Examples 4.1-4.1, Examples 5.1-5.4 and Comparative Examples 1-2 was tested using an amino acid analyzer and a fluorescent colorimetric detection method.

[0059] The test results are shown in Table 1: Table 1 Tryptophan purity test results It can be seen from Table 1 that the purity of tryptophan obtained by purification in Example 1.3 is 99.8%, indicating that the method for purifying high-purity tryptophan in Example 1.3 of the present application has a good effect on improving the purity of tryptophan.

[0060] The purity of tryptophan purified in Examples 1.1 and 1.2 of the present application is 99.4% and 99.5%, respectively; the purity of tryptophan purified in Example 1.4 of the present application is reduced by 0.7% and 0.8%, respectively, compared with Examples 1.1 and 1.2; the purity of tryptophan purified in Example 1.5 of the present application is reduced by 0.8% and 0.9%, respectively, compared with Examples 1.1 and 1.2, respectively. This indicates that when the mass ratio of gelatin, cellulose and water in step A1 is (8-10):(0.8-1.2):(135-145), the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a good effect on purifying tryptophan.

[0061] The purity of tryptophan purified in Example 1.3 of the present application is increased by 0.4% and 0.3% compared with that in Example 1.1 and Example 1.2, respectively, indicating that when the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a better effect on purifying tryptophan.

[0062] The purity of tryptophan purified in Examples 2.1 and 2.2 of the present application is 99.5% and 99.6%, respectively; the purity of tryptophan purified in Example 2.3 of the present application is reduced by 1.2% and 1.3% compared with Examples 2.1 and 2.2, respectively; the purity of tryptophan obtained in Example 2.4 of the present application is reduced by 1.1% and 1.2% compared with Examples 2.1 and 2.2, respectively, indicating that when the mass of divinylbenzene in step A2 is 0.8-0.9% of the mass of dodecylstyrene, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a good effect on purifying tryptophan.

[0063] The purity of tryptophan purified in Example 1.3 of the present application is increased by 0.3% and 0.2% compared with that in Example 2.1 and Example 2.2, respectively, indicating that when the mass ratio of gelatin, cellulose and water in Step A1 is 9:1:140 and the mass of divinylbenzene is 0.85% of the mass of dodecylstyrene, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a better effect on purifying tryptophan.

[0064] The purity of tryptophan purified in Examples 3.1 and 3.2 of the present application is 99.4% and 99.6%, respectively; the purity of tryptophan purified in Example 3.3 of the present application is reduced by 0.9% and 1.1%, respectively, compared with Examples 3.1 and 3.2; the purity of tryptophan obtained in Example 3.4 of the present application is reduced by 0.8% and 1%, respectively, compared with Examples 3.1 and 3.2, respectively, indicating that when the mass of the initiator in step A3 is 0.8%-1% of the mass of the mixture, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a good effect on purifying tryptophan.

[0065] The purity of tryptophan purified in Example 1.3 of the present application is increased by 0.4% and 0.2% compared with that in Example 3.1 and Example 3.2, respectively, indicating that when the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the mass of divinylbenzene in step A2 is 0.85% of the mass of dodecylstyrene, and the mass of the initiator in step A3 is 0.9% of the mass of the mixture, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a better effect on purifying tryptophan.

[0066] The purity of tryptophan purified in Examples 4.1 and 4.2 of the present application is 99.4% and 99.5%, respectively. The purity of tryptophan purified in Example 4.3 of the present application is reduced by 0.8% and 0.9% compared with Examples 4.1 and 4.2, respectively. The purity of tryptophan obtained in Example 4.4 of the present application is reduced by 0.7% and 0.8% compared with Examples 4.1 and 4.2, respectively. This indicates that when stirring at a speed of 80-90 r / min in Step A3, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a good effect on purifying tryptophan.

[0067] The purity of tryptophan purified in Example 1.3 of the present application is increased by 0.4% and 0.3% compared with that in Example 4.1 and Example 4.2, respectively, indicating that the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the mass of divinylbenzene in step A2 is 0.85% of the mass of dodecylstyrene, the mass of the initiator in step A3 is 0.9% of the mass of the mixture, and when stirring at a speed of 85r / min in step A3, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a better effect on purifying tryptophan.

[0068] The purity of tryptophan purified in Examples 5.1 and 5.2 of the present application was 99.3% and 99.3%, respectively. The purity of tryptophan purified in Example 5.3 of the present application was reduced by 0.7% and 0.8% compared with Examples 5.1 and 5.2, respectively. The purity of tryptophan obtained in Example 5.4 of the present application was reduced by 0.9% and 1% compared with Examples 5.1 and 5.2, respectively. This indicates that when the reaction temperature of step A4 is 80-90°C, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a good effect on purifying tryptophan.

[0069] The purity of tryptophan purified in Example 1.3 of the present application is increased by 0.3% and 0.2% compared with that in Example 5.1 and Example 5.2, respectively, indicating that the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the mass of divinylbenzene in step A2 is 0.85% of the mass of dodecylstyrene, the mass of the initiator in step A3 is 0.9% of the mass of the mixture, and when stirring at a speed of 85 r / min in step A3 and the reaction temperature in step A4 is 85°C, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a better effect on purifying tryptophan.

[0070] The purity of tryptophan purified in Comparative Examples 1 and 2 of the present application is reduced by 2.5% and 2.2% respectively compared with that in Example 1.3, indicating that the mass ratio of gelatin, cellulose and water in step A1 is 9:1:140, the mass of divinylbenzene in step A2 is 0.85% of the mass of dodecylstyrene, the mass of the initiator in step A3 is 0.9% of the mass of the mixture, and when stirring at a speed of 85r / min in step A3 and the reaction temperature in step A4 is 85°C, the obtained strongly acidic dodecylstyrene-divinylbenzene resin has a better effect on purifying tryptophan than 001×7 cation exchange resin and D061 cation exchange resin.

Claims

1. A method for purifying high-purity tryptophan, characterized in that: The method comprises the following steps: S1. microfiltration: adjusting the pH of the tryptophan fermentation broth to 3.0-3.5 with acid, filtering with a microfiltration membrane, and collecting the microfiltration clear liquid; S2. Ultrafiltration: ultrafiltration the collected microfiltration clear liquid using an ultrafiltration membrane to collect the ultrafiltration clear liquid; S3. Ion exchange resin: use ion exchange resin to adsorb tryptophan; S4. Elution: eluting with aqueous ammonia at a concentration of 2.0-2.5 mol / L, collecting the crude tryptophan extract; S5. Decolorization: decolorizing the crude tryptophan extract with activated carbon, and collecting the decolorized clear solution; S6. Crystallization: The decolorized clear solution is concentrated and crystallized under vacuum; S7. Drying: After drying, high-purity tryptophan is obtained; The ion exchange resin in step S3 is a strongly acidic dodecylstyrene-divinylbenzene resin.

2. A method for purifying high-purity tryptophan according to claim 1, characterized in that: The preparation method of the strongly acidic dodecylstyrene-divinylbenzene resin comprises the following steps: A1. Stir and mix water and gelatin evenly, react for 13-17 minutes, then add cellulose to completely dissolve it to obtain a stabilizer solution; A2. The mixture of dodecylstyrene and divinylbenzene was mixed, rinsed 2-3 times with a 4-6% sodium hydroxide solution, and then rinsed 2-3 times with water; A3. The mixture and the initiator are added to the stabilizer solution, the stabilizer solution is used as the reaction solvent, and the amount thereof is such that the mixture and the initiator can be evenly dispersed, and the mixture is heated to 60-65°C at a rate of 80-90r / min with stirring, and kept warm for 2.0-2.5h, and then heated to 70-75°C, and kept warm for 6.0-6.5h, and then heated to 85-90°C, and kept warm for 1.0-1.5h, and then filtered after cooling, rinsed and dried to obtain macroporous cross-linked styrene white balls; A4. The macroporous cross-linked styrene white ball is swollen with a swelling agent, and concentrated sulfuric acid at a concentration of 95-98% is added under stirring, and the reaction temperature is 80-90 ° C. After 10-12h, the temperature is lowered, and then the concentrated sulfuric acid is diluted to obtain a strongly acidic dodecylstyrene-divinylbenzene resin; Wherein, in the step A1, the mass ratio of gelatin, cellulose and water is (8-10):(0.8-1.2):(135-145), in the step A2, the mass of divinylbenzene is 0.8-0.9% of the mass of dodecylstyrene, and in the step A3, the mass of the initiator is 0.8%-1% of the mass of the mixture.

3. A method for purifying high-purity tryptophan according to claim 2, characterized in that: In the step A1, the mass ratio of gelatin, cellulose and water is 9:1:

140.

4. A method for purifying high-purity tryptophan according to claim 2, characterized in that: In the step A2, the mass of divinylbenzene is 0.85% of the mass of dodecylstyrene.

5. A method for purifying high-purity tryptophan according to claim 2, characterized in that: In step A3, the mass of the initiator is 0.9% of the mass of the mixture.

6. A method for purifying high-purity tryptophan according to claim 2, characterized in that: The stirring speed in step A3 is 85 r / min.

7. A method for purifying high-purity tryptophan according to claim 2, characterized in that: The reaction temperature in step A4 is 85°C.

8. A method for purifying high-purity tryptophan according to claim 1, characterized in that: In step S5, the amount of activated carbon added is 1.2-1.4%, the decolorization temperature is 55-60° C., and the decolorization time is 45-50 min.

9. A method for purifying high-purity tryptophan according to claim 1, characterized in that: The concentration end point in step S6 is 50-55 g / L.

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