Method for separating and purifying 5-hydroxytryptophan from microbial fermentation liquor

Abstract

The invention belongs to the technical field of separation and purification of fermentation products, and particularly relates to a method for separating and purifying 5-hydroxytryptophan from microbial fermentation liquor. The method comprises the steps of fermentation liquor acidification, solid-liquid separation, decoloration, concentration crystallization and macroporous resin adsorption. According to the method, the tryptophan and the 5-hydroxytryptophan in the fermentation liquor are effectively separated by using the macroporous adsorption resin, so that the problem that the 5-hydroxytryptophan is difficult to extract in microbial fermentation production is solved, the extraction process is simple to operate, few in steps and low in requirements on extraction equipment, the extraction cost is reduced, and the extraction efficiency is improved; the product extraction purity is high and is greater than 99%, the extraction yield is considerable, and the product competitiveness and the production profit are improved; the extraction process is clean and environment-friendly, so that the subsequent waste gas and waste liquid treatment cost is reduced, and the economic benefit is further improved; the high-quality, high-benefit and high-purity preparation of the 5-hydroxytryptophan is realized, and the application prospect is good.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification of fermentation products, and specifically relates to a method for separating and purifying 5-hydroxytryptophan from microbial fermentation liquid. Background Art

[0002] 5-Hydroxytryptophan (5-HTP) is a precursor for the synthesis of serotonin and melatonin in animals. 5-HTP plays an important role in regulating emotions, behaviors, sleep, pain, body temperature and other physiological functions. It is often used to treat depression, insomnia, migraine and other diseases. The large-scale production of 5-HTP will have good market prospects and industrial value.

[0003] At present, the main production routes of 5-hydroxytryptophan are natural product extraction, chemical synthesis and microbial fermentation. Among them, natural product extraction is still the main method for commercial production of 5-hydroxytryptophan. However, the method of extracting from natural plants has drawbacks that cannot be ignored. Among them, low extraction yield, susceptibility to seasonal influences, insufficient raw materials and geographical restrictions have become the main bottlenecks restricting the large-scale production of 5-HTP; the chemical method synthesis reaction system is relatively complex, the steps are cumbersome, the cost is high and the control is difficult. Microbial fermentation is low-cost and simple to operate, providing a fast and environmentally friendly alternative for the production of natural compounds with medical value. Therefore, in recent years, microbial fermentation has attracted great attention in the industry.

[0004] When microbial fermentation is used to produce 5-HTP, its separation and extraction has always been a difficult problem in the industry. Due to the immaturity of the fermentation method, the output of 5-HTP is low, the conventional extraction method has a low extraction yield, and the product loss is serious; the autolysis of the bacteria leads to an increase in soluble protein, which further increases the difficulty of separation and extraction; the fermentation by-products headed by tryptophan are difficult to control, and the physical and chemical properties of tryptophan are extremely similar to those of 5-HTP, which has become the biggest difficulty in the separation and extraction of 5-HTP. Xu Qingyang et al. (CN 113563251A) proposed using cationic resin for adsorption and then using ammonia water for elution; Zhang Shan et al.'s patent (CN 114874125A) proposed using cationic resin for adsorption and then using ethanol aqueous solution as an eluent. These solutions solve the problem of difficulty in separating tryptophan from 5-hydroxytryptophan, but the method still has two problems: on the one hand, the nature of 5-hydroxytryptophan is very unstable and is easily oxidized and blackened under alkaline conditions, and the patent with publication number CN 113563251 A uses alkaline water for elution in many places, which will affect the appearance of the finished product of 5-hydroxytryptophan; on the other hand, the patent with publication number CN114874125A uses ethanol aqueous solution as eluent, but the amount of analytical agent is large, the process stability is poor, and the separation and purification yield is low. Therefore, these problems greatly limit the application of microbial fermentation in the preparation of 5-HTP.

[0005] Therefore, exploring a method with high extraction yield and high purity for separating and purifying 5-HTP from microbial fermentation broth is an urgent problem to be solved in the art. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides a method for separating and purifying 5-hydroxytryptophan from a microbial fermentation broth, aiming at extracting 5-hydroxytryptophan from the microbial fermentation broth at low cost and high efficiency.

[0007] The present invention provides a method for separating and purifying 5-hydroxytryptophan from a microbial fermentation broth, which comprises the following steps:

[0008] Step 1, adding an antioxidant to the fermentation broth, filtering, and collecting the filtrate;

[0009] Step 2, decolorizing the filtrate collected in step 1, filtering, and collecting the filtrate;

[0010] Step 3, concentrating, crystallizing, filtering, and collecting the precipitate collected in step 2 to obtain a crude product;

[0011] Step 4, dissolving the crude product to obtain a crude product solution;

[0012] Step 5, passing the crude product solution through a macroporous adsorption resin, and collecting the effluent to obtain;

[0013] The model of the macroporous adsorption resin is selected from LXT-085, LXT-080, and LXT-036.

[0014] Preferably, the model of the macroporous adsorption resin is LXT-085.

[0015] Preferably, the antioxidant in step 1 is selected from at least one of sodium bisulfite, sodium dithionite, sodium ascorbate, and sodium metabisulfate; the mass volume ratio of the antioxidant to the fermentation broth is 0.1%-0.3%;

[0016] And / or, after adding the antioxidant in step 1, the pH is adjusted to 2.0-4.0, the reagent for adjusting the pH is sulfuric acid or acetic acid, and the mixture is allowed to stand for 10-40 minutes after the pH is adjusted;

[0017] And / or, the filtration in step 1 is performed using a ceramic membrane or a plate and frame.

[0018] Preferably, the antioxidant in step 1 is sodium dithionite, and the amount of the antioxidant is 0.1% by mass volume;

[0019] And / or, after adding the antioxidant in step 1, adjusting the pH to 2.0-3.0;

[0020] And / or, the filtration in step 1 is performed using a ceramic membrane, and the pore size of the filter element of the ceramic membrane is 30-50nm.

[0021] Preferably, the decolorization in step 2 uses activated carbon, and the amount of the activated carbon is 0.5-10% by mass volume;

[0022] And / or, the crude product dissolution in step 4 is that the crude product is dissolved in an acetic acid aqueous solution with a volume fraction of 0-5%, the concentration after dissolution is 80-140g / L, and the dissolution temperature is 20°C-60°C; and / or, after the crude product is dissolved in step 4, the pH is adjusted to 2.0-5.0, and the pH adjusting reagent used is one of acetic acid or 20% sulfuric acid.

[0023] Preferably, the concentration temperature in step 3 is 60° C.-80° C., and the concentration multiple is 3.5-8 times;

[0024] And / or, before the crystallization, the pH is adjusted to 5.0-7.0, the stirring speed during crystallization is 80-120 r / min, the temperature is 8-20° C., and the crystallization time is 2-10 hours.

[0025] Preferably, the concentration in step 3 is carried out by thin film evaporation; and / or the equipment used for the crystallization is one of HC series evaporation crystallization equipment, forced circulation evaporation crystallization equipment, DTB evaporation crystallizer, Oslo type evaporation crystallizer, and guide tube crystallization equipment;

[0026] And / or, before the crystallization in step 3, the reagent for adjusting pH is concentrated ammonia water or 10%-30% NaOH aqueous solution.

[0027] Preferably, in step 5, the flow rate of the crude product solution passing through the macroporous adsorption resin is 3-8 BV / h, the volume of the macroporous resin is 3-8 L, and the collection time is 20 min-50 min.

[0028] Preferably, after collecting the effluent in step 5, secondary crystallization may be further performed; the secondary crystallization step is to adjust the pH of the effluent to 5.0-7.0, and then gradually cool the temperature by reducing 5°C every 10 minutes to 5-10°C.

[0029] Preferably, the fermentation broth is obtained by fermentation of recombinant Escherichia coli capable of producing 5-hydroxytryptophan.

[0030] The present invention provides a method for separating and purifying 5-hydroxytryptophan from a microbial fermentation broth. The method includes fermentation broth acidification, solid-liquid separation, activated carbon decolorization, concentrated crystallization, crude product dissolution, and macroporous resin adsorption steps. The present invention optimizes various process conditions and steps, and also screens different types of macroporous resins to fill the resin column. The results show that after purification using LXT-085 macroporous resin, tryptophan is completely removed, the protein removal effect is the best, the solution transmittance and product purity are greatly improved, and the purification yield, purity and quality of 5-hydroxytryptophan can be significantly improved, the yield is greater than 80%, and the purity can reach 99.9%.

[0031] The present invention mainly solves the problems of complicated process, poor stability, low extraction yield, and difficulty in removing Trp impurities in the extraction of 5-HTP produced by microbial fermentation under low salt environment. The main advantages of this method are: (1) the extraction process is simple to operate, with few steps, and the extraction yield is improved; (2) the extraction equipment requirements are low, the equipment investment is small, the extraction cost is reduced, the extraction efficiency is improved, the purity of the obtained product is high, the main line extraction yield can reach 80%, and the product competitiveness and production profit are improved; (3) the extraction process produces less wastewater, no other solvents are introduced, clean and environmentally friendly, and has environmental friendliness, reducing the subsequent waste gas and waste liquid treatment costs, and further improving economic benefits; (4) the process uses weak acid and weak base for pH adjustment, and at the same time adopts the measure of adding antioxidants to effectively reduce the degradation rate of 5-HTP, reduce the generation of oxidized impurities in the product, further improve the quality of the product, and solve the problem of dark color of 5-HTP finished product. The method of the present invention has greatly promoted the development of microbial fermentation method for producing 5-HTP, and has good application prospects.

[0032] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0033] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The following is the extraction process flow chart;

[0035] Figure 2 This is a high performance liquid chromatogram of the related substances of 5-HTP extracted in Example 1;

[0036] Figure 3 This is a high performance liquid chromatogram of commercially available 5-HTP related substances;

[0037] Figure 4 This is the infrared spectrum of 5-HTP extracted in Example 2. DETAILED DESCRIPTION

[0038] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0039] In the following examples, the source of the recombinant Escherichia coli capable of producing 5-hydroxytryptophan is patent publication number CN116287042A.

[0040] The culture method of the fermentation broth for producing 5-hydroxytryptophan is as follows:

[0041] (1) Prepare LB solid medium. The ingredients of LB solid medium are: 10 g / L soy peptone, 5 g / L yeast extract powder, 10 g / L NaCl, and 18 g / L agarose.

[0042] (2) Activate the recombinant E. coli by streaking on LB solid medium.

[0043] (3) Pick 1-2 single colonies from the solid plate and place them in LB liquid culture medium. The composition of LB liquid culture medium is: soy peptone 12g / L, yeast extract powder 6g / L, NaCl 10g / L, ampicillin 0.1g / L. Culture in a shaking incubator at 30°C and 180rpm for 15h±2h.

[0044] (4) When OD 600When the pH value is greater than 2.0, the microorganisms are inoculated into the seed tank at an inoculation amount of 1% for seed tank culture. The seed tank culture medium is: 5 g / L yeast extract powder, 10 g / L soy peptone, 10 g / L NaCl, and 0.1 g / L ampicillin. The temperature is 30°C, the pressure is 0.040 MPa, and the stirring speed is 250 rpm.

[0045] (5) When OD 600 When the pH value is greater than 2.0, the microorganism is inoculated into the fermentation tank at an inoculation amount of 10%. The fermentation medium is an optimized LB medium: 50 g / L yeast extract, 10 g / L soy peptone, 10 g / L NaCl, 13 g / L dipotassium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 20 g / L glucose, and 40 g / L corn steep liquor. 60% (W / V) glucose is continuously added for culture.

[0046] (6) When the OD in the fermenter 600 When the pH value is ≥45, 0.15 g / L isopropyl-β-D-thiogalactoside (IPTG) is added at one time and fermented for 45-50 hours to obtain 5-hydroxytryptophan fermentation broth. The 5-hydroxytryptophan content in the fermentation broth is ≥50 g / L, and the tryptophan residue is ≤0.5 g / L.

[0047] The transmittance of the aqueous solution described in the following examples is measured by an ultraviolet spectrophotometer. The product is dissolved in water to prepare a 0.3 mol / L solution, and the transmittance is measured at 470 nm.

[0048] The crystallization equipment used in the following examples is an Oslo type crystallizer.

[0049] Example 1 A method for separating and purifying 5-hydroxytryptophan from microbial fermentation broth

[0050] This embodiment provides a method for separating and purifying 5-hydroxytryptophan from a microbial fermentation broth. Figure 1 As shown, the specific steps include the following:

[0051] (1) Microfiltration: 33.1 L of fermentation broth was inactivated and then transferred to an acid adjustment tank, sodium dithionite was added to a mass volume fraction of 0.1%. 20% sulfuric acid was used to adjust the pH to 2.80, and the mixture was allowed to stand at room temperature for 30 min. The mixture was added to a ceramic membrane (50 nm) for microfiltration, and 40.0 L of the filtrate was collected, with a 5-hydroxytryptophan content of 28.75 g / L.

[0052] (2) Activated carbon decolorization: The filtrate was transferred to a decolorization tank, and activated carbon (Jiangsu Zhuxi activated carbon) was added. The mass of activated carbon accounted for 1% of the total volume of the filtrate. Stirring and decolorizing was carried out for 30 minutes. The carbon residue was removed by plate and frame filtration. 39.8 L of decolorized liquid was collected, and the 5-hydroxytryptophan content was 26.75 g / L.

[0053] (3) Concentration and crystallization: The decolorized solution obtained in step (2) was concentrated by thin film evaporation to a 5-hydroxytryptophan concentration of 100.55 g / L. The temperature during the concentration process was controlled at 60-65° C., and the total volume of the concentrated solution was 10.0 L. The pH of the concentrated solution was adjusted to 5.53 using concentrated ammonia water, the stirring speed was reduced, and the crystals were grown for 2 h. During the crystallization process, the temperature was slowly lowered to 10.2° C. and the wet product was obtained by centrifugation. The mass of the wet product was 2262.38 g.

[0054] (4) Dissolution of wet product: Add 9 L of purified water to 2262.38 g of wet product to prepare a 90-100 g / L aqueous solution, adjust the pH to 2.84 with acetic acid, control the process temperature at 50 °C and stir until completely dissolved.

[0055] (5) Resin flow: The resin column was filled with 3.0 L of LXT-085 non-polar macroporous adsorption resin and continuously fed at a flow rate of 5 BV / h. Tryptophan and a large number of small molecular proteins, pigments and other impurities in the dissolved liquid would be adsorbed on the resin. The resin effluent was collected and the tryptophan situation was detected by TLC during the collection process. When tryptophan flowed out, the feeding was stopped. The flow time was about 30-50 min. After water top-up, a total of 12.5 L of filtrate was collected. The 5-hydroxytryptophan content in the feed liquid was 75.6 g / L.

[0056] (6) Secondary crystallization: The filtrate collected in step (5) was adjusted to pH 5.77 with concentrated aqueous ammonia, and the temperature was reduced by 5°C every 10 min to 5.8°C by gradient cooling. The filter cake was washed with 300 mL of ethyl acetate to obtain the product.

[0057] (7) Drying: The product was dried at 65°C for 6 hours to obtain 920.02 g of pure 5-hydroxytryptophan powder. The obtained product was tested by HPLC ( Figure 2 ), the content is 99.9%, and the total process yield is 80.01%.

[0058] Compared with the HPLC spectrum of commercially available 5-hydroxytryptophan ( Figure 3 ), the 5-hydroxytryptophan extracted and purified by the present invention has the same retention time and equivalent peak intensity, indicating that the purity of the 5-hydroxytryptophan extracted and purified by Example 1 reaches the level of commercially available 5-hydroxytryptophan.

[0059] Example 2 A method for separating and purifying 5-hydroxytryptophan from a microbial fermentation broth

[0060] This embodiment provides a method for separating and purifying 5-hydroxytryptophan from a microbial fermentation broth, and the specific steps include the following:

[0061] (1) Microfiltration: 35.2 L of fermentation broth was inactivated and then transferred to an acid adjustment tank. Sodium dithionite with a volume fraction of 0.1% was added and the pH was adjusted to 3.00 using 20% ​​sulfuric acid. The broth was allowed to stand at room temperature for 30 min and then microfiltered using a ceramic membrane (50 nm). 45.0 L of the filtrate was collected and the 5-hydroxytryptophan content was 26.85 g / L.

[0062] (2) Activated carbon decolorization: The filtrate was transferred to a decolorization tank, and activated carbon (Jiangsu Zhuxi activated carbon) was added. The mass of activated carbon accounted for 1% of the total volume of the filtrate. Stirring and decolorizing was carried out for 30 minutes. The carbon residue was removed by plate and frame filtration. 43.9 L of decolorized liquid was collected. The 5-hydroxytryptophan content was 26.14 g / L.

[0063] (3) Concentration and crystallization: The decolorized solution obtained in step (2) was concentrated by vacuum distillation to a 5-hydroxytryptophan concentration of 100.62 g / L. The temperature during the concentration process was controlled at 60-65° C., and the total volume of the concentrated solution was 10.5 L. The pH was adjusted to 5.52 using concentrated ammonia water, the stirring speed was reduced, and the crystals were grown for 2 h. During the crystallization process, the temperature was slowly lowered to 10.2° C., and the wet product was obtained by centrifugation. The mass of the wet product was 2377.15 g.

[0064] (4) Dissolution of wet product: Add 9 L of purified water to 2377.15 g of wet product to prepare a 90-100 g / L aqueous solution, adjust the pH to 2.84 with acetic acid, control the temperature to 55°C, raise the temperature and stir until completely dissolved to obtain a wet product solution.

[0065] (5) Resin flow: The resin column was filled with 3.0 L of LXT-085 non-polar macroporous adsorption resin. The wet product solution was continuously fed at a flow rate of 3 BV / h. Tryptophan and a large number of small molecular proteins, pigments and other impurities in the solution would be adsorbed on the resin. The resin effluent was collected. TLC was used to detect the tryptophan situation during the collection process. When tryptophan flowed out, the feeding was stopped. The flow time was about 30-50 min. After the water top was filled, a total of 12.0 L of filtrate was collected. The 5-hydroxytryptophan content in the feed liquid was 81.24 g / L.

[0066] (6) Secondary crystallization: The filtrate collected in step (5) was adjusted to pH 5.82 with concentrated aqueous ammonia, and the temperature was reduced by 5°C every 10 min to 5.6°C by gradient cooling. The filter cake was washed with 300 mL of ethyl acetate to obtain the product.

[0067] (7) Drying: The product was dried at 65°C for 6 hours to obtain 974.89 g of pure 5-hydroxytryptophan powder. The obtained product was subjected to HPLC and infrared detection (such as Figure 4 ), the content is 99.8%, and the total process yield is 80.67%.

[0068] Example 3 A method for separating and purifying 5-hydroxytryptophan from microbial fermentation broth

[0069] Separation and purification were performed according to the method described in Example 1, except that: the wet product solution obtained in step (4) was divided into three equal parts; in step (5), three different types of macroporous resins, namely LXT-085, LXT-080 and LXT-036, were respectively loaded into the resin column, and the three parts of the wet product solution were respectively passed through the three types of macroporous resins.

[0070] The solution before and after the resin flow in step (5) was tested for protein content, transmittance, 5-HTP purity, and tryptophan (Trp) content. The results are shown in the following table:

[0071] Table 1 Changes of relevant indicators before and after resin flow

[0072]

[0073] The results are shown in Table 1: After purification by LXT-085 macroporous resin, all tryptophan in the wet product solution was removed, the protein removal effect was also the best, and the solution transmittance and product purity were greatly improved, indicating that LXT-085 macroporous resin has the best purification effect on 5-hydroxytryptophan. Using LXT-085 macroporous resin to purify 5-hydroxytryptophan can significantly improve the purification efficiency, purity and quality of 5-hydroxytryptophan.

[0074] It can be seen from the above embodiments that the present invention provides a method for separating and purifying 5-hydroxytryptophan from a microbial fermentation broth. The method includes fermentation broth acidification, solid-liquid separation, activated carbon decolorization, concentrated crystallization, crude product dissolution, and resin flow steps. The present invention optimizes various process conditions and steps, and also screens different types of macroporous resins to fill the resin column. The results show that after purification using LXT-085 macroporous resin, tryptophan is completely removed, the protein removal effect is also the best, the solution transmittance and product purity are greatly improved, and the purification efficiency, purity and quality of 5-hydroxytryptophan can be significantly improved.

Claims

1. A method for separating and purifying 5-hydroxytryptophan from a microbial fermentation broth, characterized in that: The following steps are involved: Step 1, adding an antioxidant to the fermentation broth, filtering, and collecting the filtrate; Step 2, decolorizing the filtrate collected in step 1, filtering, and collecting the filtrate; Step 3, concentrating, crystallizing, filtering, and collecting the precipitate collected in step 2 to obtain a crude product; Step 4, dissolving the crude product to obtain a crude product solution; Step 5, passing the crude product solution through a macroporous adsorption resin, and collecting the effluent to obtain; The model of the macroporous adsorption resin is selected from LXT-085, LXT-080, and LXT-036.

2. The method for separating and purifying 5-hydroxytryptophan according to claim 1, characterized in that: The model of the macroporous adsorption resin is LXT-085.

3. The method for separating and purifying 5-hydroxytryptophan according to claim 1, characterized in that: The antioxidant in step 1 is selected from at least one of sodium bisulfite, sodium dithionite, sodium ascorbate, and sodium metabisulfate; the mass volume ratio of the antioxidant to the fermentation broth is 0.1%-0.3%; And / or, after adding the antioxidant in step 1, the pH is adjusted to 2.0-4.0, the reagent for adjusting the pH is sulfuric acid or acetic acid, and the mixture is allowed to stand for 10-40 minutes after the pH is adjusted; And / or, the filtration in step 1 is performed using a ceramic membrane or a plate and frame.

4. The method for separating and purifying 5-hydroxytryptophan according to claim 3, characterized in that: The antioxidant in step 1 is sodium dithionite, and the amount of the antioxidant is 0.1% by mass volume; And / or, after adding the antioxidant in step 1, adjusting the pH to 2.0-3.0; And / or, the filtration in step 1 is performed using a ceramic membrane, and the pore size of the filter element of the ceramic membrane is 30-50nm.

5. The method for separating and purifying 5-hydroxytryptophan according to claim 1, characterized in that: The decolorization in step 2 uses activated carbon, and the amount of the activated carbon is 0.5-10% by mass volume; And / or, the crude product dissolution in step 4 is that the crude product is dissolved in an acetic acid aqueous solution with a volume fraction of 0-5%, the concentration after dissolution is 80-140g / L, and the dissolution temperature is 20°C-60°C; and / or, after the crude product is dissolved in step 4, the pH is adjusted to 2.0-5.0, and the pH adjusting reagent used is one of acetic acid or 20% sulfuric acid.

6. The method for separating and purifying 5-hydroxytryptophan according to claim 1, characterized in that: The concentration temperature in step 3 is 60°C-80°C, and the concentration multiple is 3.5-8 times; And / or, before the crystallization, the pH is adjusted to 5.0-7.0, the stirring speed during crystallization is 80-120 r / min, and the temperature is 8-20° C.; The crystallization time is 2-10 hours.

7. The method for separating and purifying 5-hydroxytryptophan according to claim 6, characterized in that: The concentration in step 3 is carried out by thin film evaporation; and / or the crystallization equipment is one of HC series evaporation crystallization equipment, forced circulation evaporation crystallization equipment, DTB evaporation crystallizer, Oslo type evaporation crystallizer, and guide tube crystallization equipment; And / or, before the crystallization in step 3, the reagent for adjusting pH is concentrated ammonia water or 10%-30% NaOH aqueous solution.

8. The method for separating and purifying 5-hydroxytryptophan according to claim 1, characterized in that: In step 5, the flow rate of the crude product solution passing through the macroporous adsorption resin is 3-8 BV / h, the volume of the macroporous resin is 3-8 L, and the collection time is 20 min-50 min.

9. The method for separating and purifying 5-hydroxytryptophan according to claim 1, characterized in that: After collecting the effluent in step 5, secondary crystallization can be further performed; the secondary crystallization step is to adjust the pH of the effluent to 5.0-7.0, and then gradually cool it down by 5°C every 10 minutes to 5-10°C.

10. The method for separating and purifying 5-hydroxytryptophan according to claim 1, characterized in that: The fermentation liquid is obtained by fermenting recombinant Escherichia coli capable of producing 5-hydroxytryptophan.

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