L-tryptophan fermentation method

Abstract

The invention relates to an L-tryptophan fermentation method. The method comprises the following steps: culturing an engineering strain in a seed culture medium to prepare a seed solution; transferring the seed solution into a main fermentation culture medium; the fermentation production of the L-tryptophan is carried out by adopting a fed-batch fermentation method; after the fermentation is finished, performing multi-stage pretreatment and extraction purification on the fermentation liquor, namely performing microfiltration by adopting a microfiltration membrane to remove thalli and large-particle impurities in the fermentation liquor, so as to obtain filtrate; adjusting the pH value of the filtrate to 3.0-3.5, and removing macromolecular impurities and pigments by adopting an ultrafiltration membrane and activated carbon; carrying out nanofiltration on the filtrate by adopting a nanofiltration membrane, and / or carrying out electrodialysis combined desalination treatment on the filtrate by adopting a selective ion exchange membrane to obtain a concentrated solution rich in L-tryptophan; adjusting the pH value of the desalted concentrated solution to 5.7-6.0, carrying out accurate crystallization by adopting a programmed cooling technology and an ultrasonic-assisted nucleation technology, and carrying out secondary refined crystallization by adopting an anti-solvent crystallization method; and washing and drying the obtained crystal to obtain an L-tryptophan product.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological fermentation, and more particularly relates to a fermentation method of L-tryptophan. BACKGROUND

[0002] L-tryptophan, molecular formula C 11 H 12 N2O2, also known as L-2-amino-3-indolylpropionic acid, is one of the essential amino acids for the human body, and has a left-handed configuration.

[0003] In the food and medical fields, L-tryptophan is used as a nutritional supplement for pregnant women and special milk powder. It is also a component of amino acid infusion and can be used with vitamin B6 to improve depression, prevent and treat rough skin disease, and as a sedative for insomnia. L-tryptophan is also an important feed additive. Adding L-tryptophan to animal feed can improve the efficiency of amino acid utilization, increase feed intake, promote growth and improve immunity; it can also alleviate the stress response of piglets after weaning and improve breeding efficiency.

[0004] L-tryptophan was first obtained by hydrolysis and separation of casein, but the natural hydrolysis method for producing L-tryptophan has problems such as high cost, serious pollution and complex process. The subsequent chemical synthesis method solves some of the problems, but due to the complicated production steps of the chemical synthesis method and the greater environmental pollution, the safety of the product cannot be completely guaranteed, so a better method for more efficient production is still needed.

[0005] With the development of biotechnology, enzyme conversion, microbial conversion and microbial fermentation methods have been realized. Although there have been mature reports on enzyme conversion method for producing L-tryptophan in China, the enzyme conversion method requires a large amount of raw materials, and the substrate L-serine is expensive, almost equivalent to L-tryptophan. At the same time, the enzyme is easily inactivated in the production process, which is not conducive to large-scale production. Microbial conversion method utilizes the metabolic capacity of microorganisms to convert precursor substances into tryptophan. When the concentration of precursor substances in the conversion liquid is high, the conversion rate decreases. Moreover, the price of precursor substances is relatively high, which is not conducive to cost reduction.

[0006] Therefore, although the above two conversion methods have certain industrialization scale, they cannot effectively reduce production costs and achieve larger-scale application.

[0007] Compared with enzyme and microbial conversion methods, microbial fermentation method has the characteristics of low cost, environmentally friendly process, high cell density and high production efficiency, but there are still challenges such as long fermentation period, low product yield and conversion rate, complex extraction process and low efficiency, which limit the further improvement of its production efficiency and economic benefit. SUMMARY

[0008] The application aims to provide an L-tryptophan fermentation method with high L-tryptophan extraction rate and high purity.

[0009] The application can achieve the above-mentioned purpose by the following technical scheme. An L-tryptophan fermentation method comprises the following steps. S1, providing an L-tryptophan engineering strain; S2, culturing the engineering strain in a seed culture medium for 10-12 hours at a culture temperature of 36-38 DEG C to prepare a seed liquid; S3, transferring the seed liquid to a main fermentation culture medium with a 5-12vol% inoculation amount, wherein the main fermentation culture medium contains a carbon source, a nitrogen source, a phosphate, a sulfate and trace elements; S4, performing L-tryptophan fermentation production by using a fed-batch fermentation method; S5, after the fermentation is completed, performing multi-stage pretreatment and extraction and purification on the fermentation liquid, comprising: S51, removing bacteria and large-particle impurities in the fermentation liquid by using a microfiltration membrane with a pore size of 0.1-0.5 microns to obtain a filtrate; S52, adjusting the pH of the filtrate to 3.0-3.5, removing macromolecular impurities and pigments by using an ultrafiltration membrane with a molecular weight cut-off of 5-12 kDa and activated carbon; S53, performing nanofiltration on the filtrate by using a nanofiltration membrane, and / or performing desalination treatment on the filtrate by using a selective ion exchange membrane and electrodialysis in combination to obtain a concentrated solution rich in L-tryptophan; S54, adjusting the pH of the desalinated concentrated solution to 5.7-6.0, performing precise crystallization by using a programmed cooling technique and ultrasonic wave-assisted nucleation technique, and then performing secondary refined crystallization by using a reverse solvent crystallization method; S6, washing and drying the obtained crystal to obtain an L-tryptophan product.

[0010] Preferably, in the step S1, the engineering strain is Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli or Saccharomyces cerevisiae.

[0011] Specifically, the engineering strain is Escherichia coli W3110 or a derivative strain thereof, or Corynebacterium glutamicum ATCC13032 or a derivative strain thereof.

[0012] Preferably, in the step S1, the engineering strain is subjected to metabolic engineering modification, and the biosynthesis abilities of phosphoenolpyruvate, erythrose-4-phosphate and serine are strengthened.

[0013] Further preferably, in step S1, the metabolic engineering of the engineered strain comprises overexpression of a gene encoding a transketolase; and / or overexpression of a gene encoding a phosphoenolpyruvate synthase; and / or overexpression of a 3-phosphoglycerate dehydrogenase mutant gene that is free of L-serine feedback inhibition.

[0014] The synthesis of L-tryptophan requires two key precursors: phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), as well as the amino donor serine. Overexpression of a gene encoding a transketolase can increase the supply of E4P; overexpression of a gene encoding a phosphoenolpyruvate synthase can convert pyruvate to PEP, increasing the accumulation of PEP. Overexpression of a 3-phosphoglycerate dehydrogenase mutant gene that is free of L-serine feedback inhibition can increase the intracellular concentration of L-serine. L-serine is one of the direct precursors of tryptophan synthesis.

[0015] The present technical solution introduces a specific strong promoter sequence in the promoter region of the tryptophan synthesis-related gene, enhances the expression of the tryptophan synthesis pathway-related gene, and can make the metabolic flux of the strain flow more to the tryptophan synthesis pathway, thereby obtaining an engineered strain with high yield of tryptophan.

[0016] Further, the engineered strain is a stable expression strain without antibiotic selection marker and / or without dependence on chemical inducer.

[0017] By genome editing technology, all necessary expression modules, such as regulatory elements and coding genes, are stably integrated into the chromosome of the host strain, avoiding the use of antibiotics for plasmid selection, and eliminating the dependence on expensive and potentially toxic chemical inducers (such as IPTG). This not only reduces production costs, but also improves the genetic stability of the strain and the biosafety of the industrial production process.

[0018] Preferably, in step S3, the carbon source comprises 20-40 g / L of glucose, the nitrogen source comprises inorganic nitrogen source and organic nitrogen source; the phosphate is potassium dihydrogen phosphate or potassium hydrogen diphosphate; the sulfate is magnesium sulfate.

[0019] Further, the inorganic nitrogen source is at least one of ammonium sulfate or ammonia water; the organic nitrogen source is at least one of yeast extract, corn steep liquor, and peptone.

[0020] Further, the organic nitrogen source comprises a protein hydrolysate prepared by enzymatic hydrolysis of waste microbial protein or agricultural by-product by-product.

[0021] Further, the trace elements comprise at least one of potassium, magnesium, iron, manganese, and sodium, such as sodium chloride and ferrous sulfate.

[0022] Preferably, in step S4, the fermentation time of 0-12 hours is the cell growth stage, and the temperature is controlled at 36-38°C; the fermentation time of 12 hours to the end of fermentation is the product synthesis stage, and the temperature is controlled at 32-34°C; the rotation speed is 300-500 rpm, and the dissolved oxygen ventilation is 0.5-1.0vvm, so as to maintain the dissolved oxygen at ≥20% during the fermentation time of 0-8 hours; the rotation speed is 600-800 rpm, and the dissolved oxygen ventilation is 1.0-1.5vvm, so as to maintain the dissolved oxygen at 10-20% during the fermentation time of 8 hours to the end of fermentation.

[0023] In step S4, the fed-batch fermentation method specifically comprises: feedback control based on online monitoring of glucose concentration, when the glucose concentration is lower than 5g / L, supplementing a feed solution containing 60-80% w / v glucose, so as to maintain the glucose concentration in the fermentation broth within the range of 0.5-5g / L.

[0024] Specifically, in step S4, the fermentation time of 12 hours to the end of fermentation is generally 12-45 hours. During the whole fermentation process, the pH of the fermentation broth is 6.5-7.5.

[0025] Further, in step S4, the feed solution can further comprise concentrated complex nitrogen source, phosphate solution and trace element solution, so as to maintain the balance of nutrients during the fermentation process and avoid the exhaustion of some components as limiting factors. The concentrated complex nitrogen source can be a mixed solution of yeast extract and peptone.

[0026] Preferably, in step S51, the microfiltration membrane is a ceramic microfiltration membrane.

[0027] Preferably, in step S52, the ultrafiltration membrane is a polyether sulfone or regenerated cellulose membrane.

[0028] The ceramic microfiltration membrane is used to remove the cells and most of the suspended solids in the fermentation broth, and the ultrafiltration membrane is used to further remove macromolecular proteins, pigments and other colloidal substances, so as to reduce the burden of subsequent purification.

[0029] Preferably, in step S53, the nanofiltration membrane has a MWCO of 200-400Da, and the electric field strength of the electrodialysis is 5-15V / cm.

[0030] Specifically, in step S53, the electrodialysis mainly removes the salt ions in the feed solution by using the selective permeability of the ion exchange membrane under the action of the electric field, so as to reduce the ionic strength.

[0031] Preferably, in step S54, the cooling rate of the programmed cooling technology is 0.5-1°C / h; when the desalination and concentration liquid is cooled to near the saturation point, the ultrasonic assisted nucleation technology is performed, the ultrasonic frequency is 20-25kHz, and the power density is 0.1-0.3W / cm 2, the ultrasonic treatment time is 5-15 seconds, and 0.05-0.1% (w / w) of a crystal form regulator is added to promote the formation of the alpha crystal; in the anti-solvent crystallization method, 20-40% v / v of ethanol is added, and the specific operation is as follows: the pH of the L-tryptophan solution is adjusted to 5.5-6.2, and then ethanol is added to the L-tryptophan aqueous solution at a rate of 0.5-1.0 mL / min, while stirring at a speed of 500-100 rpm.

[0032] Preferably, in step S54, after the anti-solvent crystallization, the L-tryptophan can be further purified by a recrystallization technique, and then the crystals are collected by filtration or centrifugation.

[0033] Preferably, in step S6, the specific steps of the crystal washing and drying are as follows: a small amount of pre-cooled pure solvent is used to quickly wash the mother liquor and impurities attached to the surface of the crystals, and then the washed crystals are vacuum dried or air-dried at 50-70°C until the constant weight, to obtain the L-tryptophan product.

[0034] Specifically, the pre-cooled pure solvent is cold water, cold ethanol or L-tryptophan-poor component of the crystallization mother liquor.

[0035] The microbial fermentation method is currently the main method for the industrial production of tryptophan, which has the advantages of low cost, environmental friendliness, simple process control, reliable product quality, etc. However, the traditional purification method may have problems such as complicated operation steps, large consumption of organic solvents, environmental unfriendliness, and large loss of product in the multi-step purification process, etc., which makes it difficult to simultaneously achieve ideal levels of final extraction recovery rate and product purity. Therefore, the extraction and purification of L-tryptophan is an important part of the entire production process, and its efficiency directly affects the final product yield and quality, and significantly affects the production cost.

[0036] The beneficial effects of the present application are as follows: (1) The present application integrates the application of efficient and green extraction and purification technology to obtain high-purity L-tryptophan products, which can meet the needs of high-end application fields such as pharmaceutical raw materials, food additives and feed additives, etc., and improve the overall economic benefits. At the same time, it also has the advantages of short fermentation period, high tryptophan yield, high sugar acid conversion rate and high post-extraction rate, and can meet the requirements of different product forms for tryptophan content. DETAILED DESCRIPTION

[0037] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined invention purposes, the specific embodiments, structures, features and effects according to the present application are described in detail as follows in combination with examples.

[0038] Example 1 A fermentation method of L-tryptophan comprises the following steps: S1, the engineering strain of the present embodiment is Escherichia coli W3110, which is obtained by knocking out, integrating and replacing the promoter through the scarless gene editing technology of CRISPR-Cas9 system to obtain a high-yield L-tryptophan engineering strain; S2, the above constructed engineering strain is used for fed-batch fermentation to produce L-tryptophan in a 5L laboratory scale fermenter. The seed culture medium is LB medium containing 10 g / L of tryptone, 5 g / L of yeast extract and 10 g / L of NaCl. The strain is recovered from the glycerol preservation tube, streaked on the LB plate and cultured at 37°C for 12 hours. A single colony is picked and inoculated into 50 mL of LB liquid medium, which is cultured in a shaking flask at a temperature of 37°C and a rotation speed of 220 rpm for 10-12 hours until the optical density measured at a wavelength of 600 nanometers OD 600 reaches 2.0-3.0 to obtain the seed liquid.

[0039] S3, the seed liquid is transferred to the main fermentation medium at a 10vol% inoculation amount, and the main fermentation medium contains 30 g / L of glucose, 5 g / L of (NH4)2SO4, 2.5 g / L of KH2PO4, 1.0 g / L of MgSO4·7H2O, 10 g / L of yeast extract, 5 g / L of corn syrup (dry weight), 1 mL / L of trace element solution (containing FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O, CoCl2·6H2O, etc.), and an appropriate amount of defoaming agent.

[0040] S4, parameter control of the fermentation process: the temperature is controlled at 37°C for 0-12 hours as the bacterial growth stage, and the temperature is reduced to 32°C for 12-45 hours as the product synthesis stage. The pH is controlled at 7.0±0.2 through automatic feeding of 25% ammonia water or 2M H2SO4 throughout the process. The stirring speed is controlled at 300-500 rpm, and the aeration amount is controlled at 0.5-1.0vvm to maintain the DO not less than 20% for 0-8 hours of fermentation; the stirring speed is controlled at 600-800 rpm through linkage control, and the aeration amount is controlled at 1.0-1.5vvm to maintain the DO between 10-20% for 8-45 hours of fermentation. The feeding strategy: feedback control based on online monitoring of glucose concentration, when the glucose concentration is lower than 5 g / L, the feeding solution containing 800 g / L of sterile glucose is added to maintain the glucose concentration in the fermentation broth within the range of 0.5-5 g / L; at the same time, the concentrated nitrogen source and phosphate are added in proportion.

[0041] S5, after the fermentation is completed, the final fermentation broth volume is calculated, and the L-tryptophan concentration in the fermentation broth is detected. At the same time, the fermentation broth is subjected to multi-stage pretreatment and extraction and purification, including: S51, ceramic cross-flow microfiltration membrane with pore size of 0.22 microns is used to remove bacteria and large particulate impurities from the fermentation broth, and a clear filtrate is obtained; S52, the clear filtrate is adjusted to pH 3.0-3.5 with 2M HCl, and 2% (w / v) powdered activated carbon is added to the clear filtrate, and the mixture is stirred at 50°C for 1 hour for adsorption and decolorization, and then the activated carbon is removed by filtration. The decolorized filtrate is subjected to ultrafiltration using regenerated cellulose membrane with a molecular weight cut-off of 10 kDa to remove large molecular weight proteins and some pigments, and the ultrafiltration clear solution is collected.

[0042] S53, a chromatography column is packed with pretreated strong acid cation exchange resin, and the column volume is 10%-20% of the volume of the fermentation broth; the column is equilibrated with deionized water. The ultrafiltration clear solution is loaded into the column at a flow rate of 2-4 BV / h. After loading is completed, the column is washed with 2-3 BV of deionized water to remove unadsorbed or weakly adsorbed sugars, inorganic salts and other impurities. Then, 1M ammonia solution is used as eluent to elute the column at a flow rate of 1 BV / h, and the eluate is collected in fractions, and a 280 nm ultraviolet detector is used to monitor the elution peak of L-tryptophan. Fractions with high L-tryptophan content are combined to obtain an ammonia solution rich in L-tryptophan. The collected L-tryptophan ammonia solution is concentrated under reduced pressure on a rotary evaporator, and the water bath temperature is controlled at 50-60°C. Most of the ammonia water is removed, and the solution is concentrated to 10%-20% of the original volume, so that the L-tryptophan concentration reaches a supersaturated state.

[0043] S54, the concentrated solution is adjusted to pH 5.8-6.0 with dilute HCl or acetic acid, and the solution is slowly cooled from 50°C to 4-10°C under slow stirring, and kept at this low temperature for 6 hours to fully crystallize. The precipitated L-tryptophan crystals are collected by filtration. The crystals are washed with a small amount of pre-cooled deionized water for 2-3 times to remove impurities in the mother liquor and adhered to the surface.

[0044] S6, the washed L-tryptophan crystals are dried in a vacuum oven at 50-60°C to constant weight to obtain the L-tryptophan product.

[0045] The weight of the dried L-tryptophan product is measured, and the extraction yield (%) is calculated. The purity of the final product is detected by high performance liquid chromatography, and the L-tryptophan standard is used for quantitative determination.

[0046] Example 2 The difference between this example and Example 1 is that in step S5, the fermentation broth is subjected to multi-stage pretreatment and extraction and purification as follows: S51, a high-speed centrifuge is used, and the speed is adjusted to 8000 rpm, and the centrifugation time is 15 minutes, and the supernatant is collected; then, the supernatant is filtered through a 0.45 micron microporous filter to obtain a clear filtrate.

[0047] S52, the pH of the clarified filtrate was adjusted to 3.0 with 2M HCl, and the solution was loaded onto a strong acid cation exchange resin column, type Amberlite IR120H, previously equilibrated with a buffer at pH 3.0 + The column volume was 1.5 L, and the loading flow rate was controlled at 2 column bed volumes per hour. After loading, the unabsorbed impurities were washed away with 5 column bed volumes of buffer at pH 3.0, and then a gradient elution was performed with a buffer at pH 5.0 containing 0.5 M NaCl, and the elution peak fractions of L-tryptophan were collected. The elution peak fractions of L-tryptophan were heated to 50°C, and 2% of the mass of the elution peak fractions of L-tryptophan was added as powdered activated carbon, and stirred for 1 hour for adsorption. After adsorption, the activated carbon was separated by a microporous filter. The decolorized filtrate was further subjected to a polyether sulfone membrane with a molecular weight cut-off of 10 kDa to remove macromolecular proteins and part of the pigments, and the ultrafiltration clear solution was collected.

[0048] The remaining components, preparation steps and parameters were the same.

[0049] Example 3 The difference between this example and Example 1 is that the engineering strain used in this example is the genetically engineered Corynebacterium glutamicum TRP-M5.

[0050] The step of step S52 is that the clarified filtrate is first subjected to a polyether sulfone membrane with a molecular weight cut-off of 10 kDa to remove residual proteins, polysaccharides and other macromolecular impurities. Then, 1% of the weight of the dry matter of the filtrate is added as granular activated carbon, and stirred at 50°C for 30 minutes for decolorization, and then filtered to remove the activated carbon.

[0051] The step of step S53 is that the ultrafiltration permeate is further subjected to a polyamide composite nanofiltration membrane with a molecular weight cut-off of 200 Da, and concentrated and partially desalted at an operating pressure of 1.5 MPa, while L-tryptophan is effectively retained. The nanofiltration retention concentrate is collected.

[0052] The remaining components, preparation steps and parameters were the same.

[0053] The post-extraction yield results and purity of the products of Examples 1-3 are shown in Table 1.

[0054] Table 1 As can be seen from the test results in Table 1, using the purification process proposed in the present application, L-tryptophan in the fermentation broth can be efficiently recovered, and the post-extraction yield can reach more than 80%, and the content of L-tryptophan in the obtained pure product is all ≥98%, which is significantly better than the prior art, and has outstanding technical effects and industrial application prospects.

[0055] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content to obtain equivalent embodiments with equivalent changes, as long as the changes or modifications do not deviate from the technical solution of the present application. Any modification, change, equivalent change and modification of the above embodiments made according to the technical essence of the present application still belong to the scope of the technical solution of the present application.

Claims

1. An L-tryptophan fermentation method, characterized in that: The following steps are involved: S1. Provide an L-tryptophan engineered strain; S2. Cultivating the engineered strain in a seed culture medium for 10-12 hours at a culture temperature of 36-38° C. to prepare a seed solution; S3, transferring the seed solution to the main fermentation medium at an inoculum rate of 5-12 vol%, wherein the main fermentation medium contains a carbon source, a nitrogen source, phosphate, sulfate and trace elements; S4, using a fed-batch fermentation method to produce L-tryptophan; S5. After the fermentation is completed, the fermentation liquid is subjected to multi-stage pretreatment and extraction and purification, including: S51, using a microfiltration membrane with a pore size of 0.1-0.5 micron to remove bacteria and large particles of impurities in the fermentation broth to obtain a filtrate; S52, adjusting the pH of the filtrate to 3.0-3.5, and removing macromolecular impurities and pigments using an ultrafiltration membrane with a molecular weight cutoff of 5-12 kDa and activated carbon; S53, performing nanofiltration on the filtrate using a nanofiltration membrane, and / or performing electrodialysis combined with desalination on the filtrate using a selective ion exchange membrane, to obtain a concentrated solution rich in L-tryptophan; S54, adjusting the pH of the concentrate to 5.7-6.0, using programmed cooling technology and ultrasonic-assisted nucleation technology to perform precise crystallization, and then performing secondary refined crystallization by anti-solvent crystallization method; S6. Washing and drying the obtained crystals to obtain L-tryptophan product.

2. An L-tryptophan fermentation method according to claim 1, characterized in that, In step S1, the engineered strain is Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli or Saccharomyces cerevisiae.

3. The L-tryptophan fermentation method according to claim 1, wherein In step S3, the carbon source includes 20-40 g / L of glucose, the nitrogen source includes an inorganic nitrogen source and an organic nitrogen source; the phosphate is potassium dihydrogen phosphate or potassium dihydrogen phosphate; and the sulfate is magnesium sulfate.

4. The L-tryptophan fermentation method according to claim 1, wherein In step S4, the fermentation time is 0-12 hours, which is the bacterial growth stage, and the temperature is controlled at 36-38°C; the fermentation time is 12 hours to the end of the fermentation, which is the product synthesis stage, and the temperature is controlled at 32-34°C. During the fermentation time, the rotation speed is 300-500 rpm, the dissolved oxygen ventilation volume is 0.5-1.0 vvm, and the dissolved oxygen is maintained at ≥20% during the fermentation time, and the rotation speed is 600-800 rpm, the dissolved oxygen ventilation volume is 1.0-1.5 vvm, and the dissolved oxygen is maintained at 10-20% during the fermentation time.

5. The L-tryptophan fermentation method according to claim 1, wherein In step S4, the fed-batch fermentation method is specifically as follows: based on feedback control of online monitoring of glucose concentration, when the glucose concentration is lower than 5 g / L, a feed solution containing 60-80% w / v glucose is added to maintain the glucose concentration in the fermentation broth in the range of 0.5-5 g / L.

6. The L-tryptophan fermentation method according to claim 1, wherein In step S52, the ultrafiltration membrane is a polyethersulfone or a regenerated cellulose membrane.

7. The L-tryptophan fermentation method according to claim 1, wherein In step S53, the MWCO of the nanofiltration membrane is 200-400 Da, and the electric field strength of the electrodialysis is 5-15 V / cm.

8. The L-tryptophan fermentation method according to claim 1, wherein In step S54, the cooling rate of the programmed cooling technology is 0.5-1°C / h; when the desalted concentrate is cooled to near the saturation point, the ultrasonic assisted nucleation technology is performed, the ultrasonic frequency is 20-25kHz, and the power density is 0.1-0.3W / cm 2 The ultrasonic treatment time is 5-15 seconds, and 0.05-0.1% (w / w) crystal form regulator is added to promote the formation of α-type crystals. The solvent added in the anti-solvent crystallization method is 20-40% v / v ethanol. The specific operation is: adjusting the pH of the L-tryptophan solution to 5.5-6.2, and then adding ethanol to the L-tryptophan aqueous solution at a rate of 0.5-1.0 mL / min, while stirring at a speed of 500-100 rpm.

9. The L-tryptophan fermentation method according to claim 1, wherein In step S6, the specific steps of washing and drying the crystals are: quickly washing the mother liquor and impurities attached to the surface of the crystals with a small amount of pre-cooled pure solvent, and then vacuum drying or air flow drying the washed crystals at 50-70° C. until constant weight is obtained to obtain L-tryptophan product.

10. The L-tryptophan fermentation method according to claim 9, characterized in that: The pre-cooled pure solvent is cold water, cold ethanol or the L-tryptophan-poor component of the crystallization mother liquor.

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