Method for producing L-histidine through fermentation

Abstract

The invention belongs to the technical field of biological fermentation, and discloses a method for producing L-histidine through fermentation, which comprises the following steps: step 1) preparing a primary seed solution, step 2) preparing a secondary seed solution, step 3) fermenting, step 4) centrifuging and filtering, step 5) decoloring, and step 6) crystallizing. In the separation and purification process, ion exchange resin is prevented from being used for separation, the amount of generated wastewater is small, and the method is environment-friendly; the method mainly adopts the processes of centrifugation, filtration, decoloration and the like, avoids the use of organic solvents, is simple and feasible to operate, and is suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a method for producing L-histidine by fermentation. Background Art

[0002] Histidine (L-Histidine), chemical name α-amino β-imidazolyl propionic acid, is a basic amino acid containing an imidazole nucleus in the molecule. L-Histidine is a semi-essential amino acid for the human body and is widely used in the pharmaceutical, food and feed industries, as well as in the production of tobacco products and cosmetics. The production of L-histidine is mainly based on protein hydrolysis, but this method has problems such as cumbersome operation steps, large environmental pollution, and high production costs. In recent years, microbial fermentation has been successfully applied to the industrial production of a variety of amino acids. It has the advantages of short fermentation cycle, low cost, mild reaction conditions, easy process control and high yield. It is also the preferred method for industrial production of L-histidine. The production of L-histidine by microbial fermentation has broad prospects, and its yield still has a lot of room for improvement.

[0003] The existing technology for histidine biofermentation research mainly includes factors such as screening strains, optimizing culture processes and adding stimulating factors.

[0004] Literature 1: Biotechnology In 2008, Zhao Jianyun et al. found that adding calcium gluconate to the fermentation medium can increase the flux of the HMP pathway and is beneficial to the synthesis of histidine in Corynebacterium glutamicum CLW0911.

[0005] Reference 2: Microbial Technology Communications In 2009, Zhu Wenzhe et al. provided a method for improving the yield of L-histidine fermentation process. Based on the principle of adjusting the distribution of cell metabolic flux, the method of adding citric acid or its salt to the culture medium was used to effectively improve the yield of L-histidine fermentation in Corynebacterium glutamicum TL1105.

[0006] Document 3: CN112481325A, the acid production mechanism and tolerance to stimulation factors of different strains vary greatly, but it is not of reference significance. Although the prior art has made a lot of research on the acid production mechanism of glutamate, for example, adding an appropriate amount of sodium malate to the fermentation medium can increase the amount of histidine produced by Corynebacterium glutamicum, but sodium malate has no obvious stimulating effect on Serratia marcescens ATCC31026; and the ability of Serratia marcescens to produce histidine is significantly improved by adding calcium acetate and malonic acid.

[0007] The pathways of energy and material metabolism in prokaryotic cells are a very complex network. It is still not clear how changing genes in the metabolic pathways will affect the energy and material flow. Since the metabolic pathways of different strains are also quite different, different fermentation strains need to be tested by experimental means. How to improve the fermentation performance of specific strains is a technical problem that needs to be solved.

[0008] In response to the above problems, the applicant's previous patent technology "CN2024111171676" provides a process for producing L-histidine by biological fermentation, which regulates the synthesis of L-histidine by adjusting the metabolic network. Based on the above research, the present invention continues to separate and extract the fermentation broth and optimize its process. Summary of the invention

[0009] On the basis of existing research, the present invention provides a method for producing L-histidine by fermentation, the fermentation efficiency of the process is high, the separation and purification process is simple and feasible, and the yield and purity of the prepared product are improved.

[0010] Aromatic or heterocyclic amino acids including histidine, tryptophan, valine and phenylalanine are all related to the HMP pathway. Erythrose-4-phosphate in the pathway is a key raw material for the synthesis of aromatic and heterocyclic amino acids (phenylalanine, tyrosine, tryptophan and histidine). The tryptophan production capacity of Escherichia coli TR2023D-1 has increased significantly compared with the starting strain, indicating that the metabolic flow of the HMP pathway has increased, and the synthesis capacity of other amino acids related to the HMP pathway (including histidine) is also likely to be upregulated. This strain is a phenylalanine and tyrosine deficient strain, and there is no need to regulate the synthesis of phenylalanine and tyrosine.

[0011] Based on the above theory, the histidine production performance of tryptophan-producing Escherichia coli TR2023D-1 was studied and optimized. The strain was deposited in the General Microbiological Center of China Culture Collection Administration with the deposit number CGMCC No.27549 (CN117778225A, already deposited).

[0012] The present invention is achieved through the following technical solutions.

[0013] A method for producing L-histidine by fermentation, comprising the following steps: Step 1) preparing a primary seed solution, step 2) preparing a secondary seed solution, step 3) fermenting, step 4) centrifuging and filtering, step 5) decolorizing, and step 6) crystallizing.

[0014] Specifically, the method comprises the following steps: Step 1) preparing a primary seed solution: activating the L-histidine-producing Escherichia coli, inoculating it into LB liquid culture medium, and culturing it at 37° C. with shaking for 36 hours to obtain a primary seed solution; Step 2) Prepare the secondary seed solution: inoculate the primary seed solution into the secondary seed medium at a rate of 15%, and culture at 37°C with shaking until the OD 600 When the value is about 8, the secondary seed solution is obtained; Step 3) Fermentation: The secondary seed liquid is inoculated at 8% into an automatic control fermenter containing a fermentation medium, the culture temperature is 36° C., the reducing sugar content is maintained at 5 g / L by feeding a glucose solution, and the feeding of the glucose solution is stopped 4 hours before the end of the fermentation; the pH in the fermenter is maintained at 7.0 by feeding 25% ammonia water, and a defoaming agent is added to defoam; the total fermentation time is 48 hours, and L-histidine fermentation liquid is obtained; Step 4) Centrifugation and filtration: The L-histidine fermentation broth enters a disc centrifuge for centrifugation, the upper layer of liquid is collected, and then filtered through a ceramic membrane to remove large molecular proteins, the filtrate is collected, and then ultrafiltration is performed through an ultrafiltration membrane to collect the ultrafiltrate; Step 5) Decolorization: Add activated carbon to the ultrafiltrate at a ratio of 1g:100ml, control the temperature at 75°C, stir and decolorize for 60 minutes, then let it stand for 30 minutes, remove the activated carbon by plate and frame filtration, and collect the filtrate; Step 6) Crystallization: Sodium hydroxide is added to the filtrate to adjust the pH to 7.6, and then the temperature is cooled at a cooling rate of 10°C / h, and the cooling crystallization is carried out by stirring at 100 rpm. When the temperature drops to 5°C, the stirring is stopped, and the crystallization is allowed to stand for 2 hours, and then the wet crystals are separated by centrifugation and vacuum dried at 70°C to obtain the L-histidine product.

[0015] Preferably, the secondary seed culture medium components are: 30 g / L sucrose, 5 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate, 5 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 50 mg / L ferrous sulfate heptahydrate, and 0.1 mg / L biotin; and the pH is controlled at 7.0.

[0016] Preferably, the fermentation medium components are: 60 g / L glucose, 10 g / L corn steep liquor, 8 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 2 g / L citric acid, 1.5 g / L magnesium sulfate heptahydrate, 0.4 g / L sodium malate, 100 mg / L ferrous sulfate heptahydrate, 10 mg / L indolepropionic acid, 0.5 mg / L thiamine, and 0.2 mg / L biotin; the pH is controlled at 7.0.

[0017] Preferably, the components of the glucose solution are: 500 g / L glucose + 15 g / L adenosine monophosphate.

[0018] Preferably, the molecular weight cut-off of the ceramic membrane is 10000 Da.

[0019] Preferably, the molecular weight cut-off of the ultrafiltration membrane is 500Da.

[0020] Preferably, the disc centrifuge is centrifuged at 5000 rpm for 3 min.

[0021] The beneficial effects achieved by the present invention mainly include but are not limited to the following aspects: The metabolic network is a very complex multi-enzyme synchronous catalytic reaction system. Only changing one of the rate-limiting reactions may not have a great impact on product synthesis. Therefore, to optimize a certain metabolic network, it is necessary to simultaneously adjust the main nodes that affect the product so that the metabolic flux can be redistributed to facilitate the accumulation of the target product. The present invention greatly improves the histidine production performance of tryptophan-producing Escherichia coli TR2023D-1 by optimizing conditions such as fermentation medium and stimulating factors, and correspondingly weakens the synthesis efficiency of tryptophan in the HMP pathway. Malate can inhibit the activity of pyruvate kinase, reduce the accumulation of pyruvate, and cause the accumulation of phosphoenolpyruvate. Phosphoenolpyruvate is an inhibitor of phosphofructokinase, the rate-limiting enzyme of the EMP pathway, so the accumulation of phosphoenolpyruvate reduces the activity of phosphofructokinase, thereby reducing the flux of the EMP pathway and alleviating the metabolic overflow between EMP and TCA. At the same time, the total glucose flux does not change, so more glucose enters the HMP synthesis pathway, and the yield of L-histidine is correspondingly increased.

[0022] Anthranilate synthase is the key enzyme in tryptophan synthesis. Indolepropionic acid, as an allosteric inhibitor of anthranilate synthase, weakens the activity of the enzyme and reduces the anabolic flux of tryptophan in the HMP pathway to increase the metabolic flux in the direction of histidine synthesis. Adenosine monophosphate (AMP) is the direct precursor of ATP synthesis, which may improve the efficiency of histidine synthesis by synthesizing ATP or activating key enzymes in histidine synthesis.

[0023] In the separation and purification process of the present invention, the use of ion exchange resin for separation is avoided, the amount of waste water generated is small, and it is environmentally friendly; the present invention mainly avoids the use of organic solvents through processes such as centrifugation, filtration and decolorization, and the operation is simple and feasible, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :Effect of indolepropionic acid on histidine production by Escherichia coli TR2023D-1.

[0025] Figure 2 :Effect of adenosine monophosphate on histidine production by Escherichia coli TR2023D-1. DETAILED DESCRIPTION

[0026] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products and methods of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products and methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments. Example

[0027] Histidine fermentation process.

[0028] Escherichia coli TR2023D-1 was activated, inoculated into LB liquid medium, and cultured at 37°C with shaking for 36 hours to obtain a primary seed solution; The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 15%, and cultured at 37°C with shaking until the OD 600 When the value is about 8, the secondary seed solution is obtained; Secondary seed culture medium components: sucrose 30 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 5 g / L, yeast extract 5 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 50 mg / L, biotin 0.1 mg / L; pH was controlled at 7.0.

[0029] The secondary seed liquid was inoculated at 8% into an automatic control fermentation tank containing a fermentation medium, the culture temperature was 36°C, the dissolved oxygen content was 20%, and the reducing sugar content was maintained at 5g / L by feeding a glucose solution (components: glucose 500g / L + adenosine monophosphate 15g / L), and the feeding of the glucose solution was stopped 4 hours before the end of the fermentation; the pH in the fermentation tank was maintained at 7.0 by feeding 25% ammonia water, and a defoaming agent was added to defoam; the fermentation time was 48 hours, and L-histidine fermentation liquid was obtained; Fermentation medium components: glucose 60g / L, corn steep liquor 10g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 5g / L, citric acid 2g / L, magnesium sulfate heptahydrate 1.5g / L, sodium malate 0.4g / L, ferrous sulfate heptahydrate 100mg / L, indolepropionic acid 10mg / L, thiamine 0.5mg / L, biotin 0.2mg / L; pH was controlled at 7.0. Example

[0030] Histidine fermentation process.

[0031] Escherichia coli TR2023D-1 was activated, inoculated into LB liquid medium, and cultured at 37°C with shaking for 36 hours to obtain a primary seed solution; The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 15%, and cultured at 37°C with shaking until the OD 600 When the value is about 8, the secondary seed solution is obtained; Secondary seed culture medium components: sucrose 30 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 5 g / L, yeast extract 5 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 50 mg / L, biotin 0.1 mg / L; pH was controlled at 7.0.

[0032] The secondary seed liquid was inoculated at 8% into an automatic control fermentation tank containing a fermentation medium, the culture temperature was 36°C, the dissolved oxygen content was 20%, and the reducing sugar content was maintained at 5g / L by feeding a glucose solution (components: glucose 600g / L + adenosine monophosphate 20g / L), and the feeding of the glucose solution was stopped 4 hours before the end of the fermentation; the pH in the fermentation tank was maintained at 7.0 by feeding 25% ammonia water, and a defoaming agent was added to defoam; the fermentation time was 50 hours, and L-histidine fermentation liquid was obtained; Fermentation medium components: glucose 60g / L, corn steep liquor 10g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 5g / L, citric acid 2g / L, magnesium sulfate heptahydrate 1.5g / L, sodium malate 0.3g / L, ferrous sulfate heptahydrate 100mg / L, indolepropionic acid 15mg / L, thiamine 0.5mg / L, biotin 0.2mg / L; pH was controlled at 7.0.

[0033] Comparative Example 1 Histidine fermentation process.

[0034] Escherichia coli TR2023D-1 was activated, inoculated into LB liquid medium, and cultured at 37°C with shaking for 36 hours to obtain a primary seed solution; The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 15%, and cultured at 37°C with shaking until the OD 600 When the value is about 8, the secondary seed solution is obtained; Secondary seed culture medium components: sucrose 30 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 5 g / L, yeast extract 5 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 50 mg / L, biotin 0.1 mg / L; pH was controlled at 7.0.

[0035] The secondary seed liquid was inoculated at 8% into an automatic control fermenter containing a fermentation medium, the culture temperature was 36°C, the dissolved oxygen content was 20%, and the reducing sugar content was maintained at 5g / L by adding a glucose solution (composition: 500g / L glucose), and the addition of the glucose solution was stopped 4 hours before the end of the fermentation; the pH in the fermenter was maintained at 7.0 by adding 25% ammonia water, and a defoaming agent was added to defoam; the fermentation time was 48 hours, and L-histidine fermentation liquid was obtained; Fermentation medium components: glucose 60g / L, corn steep liquor 10g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 5g / L, citric acid 2g / L, magnesium sulfate heptahydrate 1.5g / L, ferrous sulfate heptahydrate 100mg / L, thiamine 0.5mg / L, biotin 0.2mg / L; pH was controlled at 7.0.

[0036] Comparative Example 2 Histidine fermentation process.

[0037] Escherichia coli TR2023D-1 was activated, inoculated into LB liquid medium, and cultured at 37°C with shaking for 36 hours to obtain a primary seed solution; The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 15%, and cultured at 37°C with shaking until the OD 600 When the value is about 8, the secondary seed solution is obtained; Secondary seed culture medium components: sucrose 30 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 5 g / L, yeast extract 5 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 50 mg / L, biotin 0.1 mg / L; pH was controlled at 7.0.

[0038] The secondary seed liquid was inoculated at 8% into an automatic control fermenter containing a fermentation medium, the culture temperature was 36°C, the dissolved oxygen content was 20%, and the reducing sugar content was maintained at 5g / L by adding a glucose solution (composition: 500g / L glucose), and the addition of the glucose solution was stopped 4 hours before the end of the fermentation; the pH in the fermenter was maintained at 7.0 by adding 25% ammonia water, and a defoaming agent was added to defoam; the fermentation time was 48 hours, and L-histidine fermentation liquid was obtained; Fermentation medium components: glucose 60g / L, corn steep liquor 10g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 5g / L, citric acid 2g / L, magnesium sulfate heptahydrate 1.5g / L, sodium malate 0.4g / L, ferrous sulfate heptahydrate 100mg / L, thiamine 0.5mg / L, biotin 0.2mg / L; pH was controlled at 7.0. Example

[0039] By optimizing the fermentation metabolic flux, the changes in the content of histidine and tryptophan in the fermentation broth obtained from different examples were compared. See Table 1 below for details.

[0040] Table 1 Examples Histidine g / L Tryptophan g / L Example 1 49.8 16.5 Example 2 48.7 17.1 Comparative Example 1 16.3 29.2 Comparative Example 2 20.6 33.4 As shown in Table 1 above, on the basis of Comparative Example 1, the addition of sodium malate in Comparative Example 2 can enhance the carbon source metabolic flow in the HMP pathway, thereby facilitating the synthesis of histidine and tryptophan; Example 1-2 significantly reduces the production of tryptophan by weakening the tryptophan synthesis pathway and strengthening the histidine synthesis pathway, and accordingly, the production of histidine is significantly increased. Example

[0041] 1. Based on Comparative Example 2, the effect of the amount of indolepropionic acid added to the culture medium on the yield of histidine produced by Escherichia coli TR2023D-1 fermentation was studied. The amount of indolepropionic acid added was set to 0, 5, 10, 20, 40, 80, with a total of six concentration gradients, in mg / L. Figure 1 As shown in the figure, the addition of low concentration of indolepropionic acid can significantly increase the production of histidine. When the addition of indolepropionic acid increases to 20 mg / L, the production of histidine decreases. The possible reason is that the enzymes related to tryptophan synthesis cannot be excessively weakened, otherwise it will lead to a decrease in the activity of Escherichia coli TR2023D-1, thereby causing a decline in acid production capacity.

[0042] 2. The amount of indolepropionic acid added to the fermentation medium was selected as 10 mg / L, and the effect of adding adenosine monophosphate to the glucose nutrient solution on the production of histidine by Escherichia coli TR2023D-1 was further studied. The amount of adenosine monophosphate added was set to 0, 5, 10, 15, 20, 25, 30, seven gradient concentrations, in g / L. Figure 2 As shown, adenosine monophosphate is positively correlated with the efficiency of histidine production by Escherichia coli TR2023D-1 fermentation. When the addition amount of adenosine monophosphate reaches 15 g / L, the production of histidine remains stable.

[0043] 3. The above study shows that when the amount of indolepropionic acid added to the fermentation medium is 10 mg / L and the concentration of adenosine monophosphate in the nutrient solution is 15 g / L, the amount of histidine produced by Escherichia coli TR2023D-1 fermentation is 49.8 g / L, while the yield when indolepropionic acid and adenosine monophosphate are not added is 20.6 g / L (Comparative Example 2), and the yield when only indolepropionic acid is added is 32.9 g / L ( Figure 1 ), the yield when only adenosine monophosphate was added was 28.5 g / L.

[0044] The present invention also verifies the effect of the above-mentioned optimal addition amount of indolepropionic acid and adenosine monophosphate on Serratia marcescens ATCC31026, and does not significantly increase the production of histidine, which also verifies the conclusion that the acid production mechanism and tolerance to stimulation factors of different strains vary greatly. Example

[0045] Separation and purification process of histidine Taking the L-histidine fermentation broth obtained in Example 1 as an example, the fermentation broth enters a disc centrifuge and is centrifuged at 5000 rpm for 3 min, the upper liquid is collected, and then filtered through a ceramic membrane (molecular weight cutoff of 10000 Da) to remove macromolecular proteins, the filtrate is collected, and then ultrafiltration is performed through an ultrafiltration membrane (molecular weight cutoff of 500 Da), and the ultrafiltrate is collected; Add activated carbon to the ultrafiltrate at a ratio of 1g:100ml, control the temperature at 75°C, stir and decolorize for 60min, then let stand for 30min, remove the activated carbon by plate and frame filtration, and collect the filtrate; Sodium hydroxide was added to the filtrate to adjust the pH to 7.6, and then the temperature was lowered at a rate of 10°C / h, and the temperature was stirred at 100rpm for crystallization. When the temperature dropped to 5°C, the stirring was stopped, and the crystallization was allowed to stand for 2 hours, and then the wet crystals were separated by centrifugation and dried in a vacuum oven at 70°C to obtain the L-histidine product. After testing, the purity of L-histidine was 95.3%, and the yield was 72.6%.

[0046] Although the above describes the specific implementation of the present invention in combination with the embodiments, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for producing L-histidine by fermentation, comprising the following steps: Step 1) preparing a primary seed solution, step 2) preparing a secondary seed solution, step 3) fermenting, step 4) centrifuging and filtering, step 5) decolorizing, and step 6) crystallizing.

2. The method according to claim 1, characterized in that The method comprises the following steps: Step 1) preparing a primary seed solution: activating the L-histidine-producing Escherichia coli, inoculating it into LB liquid culture medium, and culturing it at 37° C. with shaking for 36 hours to obtain a primary seed solution; Step 2) Prepare the secondary seed solution: inoculate the primary seed solution into the secondary seed medium at a rate of 15%, and culture at 37°C with shaking until the OD 600 When the value is about 8, the secondary seed solution is obtained; Step 3) Fermentation: The secondary seed liquid is inoculated at 8% into an automatic control fermenter containing a fermentation medium, the culture temperature is 36° C., the reducing sugar content is maintained at 5 g / L by feeding a glucose solution, and the feeding of the glucose solution is stopped 4 hours before the end of the fermentation; the pH in the fermenter is maintained at 7.0 by feeding 25% ammonia water, and a defoaming agent is added to defoam; the total fermentation time is 48 hours, and L-histidine fermentation liquid is obtained; Step 4) Centrifugation and filtration: The L-histidine fermentation broth enters a disc centrifuge for centrifugation, the upper layer of liquid is collected, and then filtered through a ceramic membrane to remove large molecular proteins, the filtrate is collected, and then ultrafiltration is performed through an ultrafiltration membrane to collect the ultrafiltrate; Step 5) Decolorization: Add activated carbon to the ultrafiltrate at a ratio of 1g:100ml, control the temperature at 75°C, stir and decolorize for 60 minutes, then let it stand for 30 minutes, remove the activated carbon by plate and frame filtration, and collect the filtrate; Step 6) Crystallization: Sodium hydroxide is added to the filtrate to adjust the pH to 7.6, and then the temperature is cooled at a cooling rate of 10°C / h, and the cooling crystallization is carried out by stirring at 100 rpm. When the temperature drops to 5°C, the stirring is stopped, and the crystallization is allowed to stand for 2 hours, and then the wet crystals are separated by centrifugation and vacuum dried at 70°C to obtain the L-histidine product.

3. The method according to claim 2, characterized in that The secondary seed culture medium comprises: 30 g / L sucrose, 5 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate, 5 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 50 mg / L ferrous sulfate heptahydrate, and 0.1 mg / L biotin; and the pH is controlled at 7.

0.

4. The method according to claim 2, characterized in that: The fermentation medium comprises: 60 g / L glucose, 10 g / L corn steep liquor, 8 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 2 g / L citric acid, 1.5 g / L magnesium sulfate heptahydrate, 0.4 g / L sodium malate, 100 mg / L ferrous sulfate heptahydrate, 10 mg / L indolepropionic acid, 0.5 mg / L thiamine and 0.2 mg / L biotin; the pH value is controlled at 7.

0.

5. The method according to claim 2, characterized in that: The components of the glucose solution are: 500 g / L glucose+15 g / L adenosine monophosphate.

6. The method according to claim 2, characterized in that The molecular weight cut-off of the ceramic membrane is 10000Da.

7. The method according to claim 2, characterized in that The molecular weight cut-off of the ultrafiltration membrane is 500Da.

8. The method according to claim 2, characterized in that: The disc centrifuge was centrifuged at 5000 rpm for 3 min.

9. The L-histidine product obtained according to the method of claims 1-8.

10. Use of the L-histidine product according to claim 9 in the preparation of medicines.

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