Refining production method of histidine

Abstract

The invention discloses a refined production method of histidine, which comprises the following steps: preparing a corynebacterium glutamicum seed solution, inoculating the corynebacterium glutamicum seed solution into a fermentation tank containing a fermentation culture medium, and fermenting to obtain a fermentation solution; filtering the fermentation liquor, concentrating, cleaning and drying to obtain a histidine crude product; adding pure water into the histidine crude product, heating and dissolving; after dissolving, adding activated carbon for adsorption and decoloration; and decolorizing, adsorbing by using acidic ion exchange resin, eluting by using ammonia water, concentrating and crystallizing the eluent, cooling and drying to obtain a histidine refined product. According to the method, the histidine crude product is treated and purified by using the acidic ion exchange resin, and is concentrated and refined in a crystallization manner, so that the high-purity histidine refined product is obtained.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of amino acid production processes, in particular to a refined production method of histidine. BACKGROUND

[0002] Histidine is an alpha-amino acid, which is mainly extracted from pig blood, cow blood or defatted soybean hydrolysate, and is obtained through ion exchange resin separation, concentration crystallization and other processes in industrial production. As a semi-essential amino acid, L-histidine is an important component of amino acid infusion and compound amino acid preparation, and is applied to the fields of nutritional fortification, gastric ulcer treatment and biochemical research. The imidazole group in the side chain has proton transfer characteristics, and is often used as a key structure of enzyme active center. The metabolic pathway includes deamination and decarboxylation to form histamine. It is particularly important for the growth and development of infants and is widely present in meat, dairy products and some plants.

[0003] The central metabolic pathway of L-histidine production bacteria corynebacterium glutamicum includes glycolysis pathway (EMP), tricarboxylic acid cycle (TCA cycle) and pentose phosphate pathway (HMP). The TCA cycle is the main oxidation pathway in the fermentation process of the genus glutamicum, and the reaction catalyzed by phosphoenolpyruvate carboxylase is the main backfilling reaction of the TCA cycle.

[0004] Document 1: CN112592941A A method for reducing the viscosity of L-histidine fermentation broth Document 1 discloses that in order to solve the problem of thick liquid in the fermentation process, high-concentration potassium chloride is added to improve the osmotic liquid of the bacterial body, and the addition of trypsin can not only reduce the content of extracellular protein polysaccharide in the fermentation broth, reduce the viscosity and improve the dissolved oxygen, but also hydrolyze the protein to generate various amino acids which can be utilized by the bacterial body again, thereby effectively improving the L-histidine fermentation unit.

[0005] In document 1, Serratia marcescens is used to produce L-histidine by fermentation. During the fermentation process, as the bacterial concentration increases, the bacterial body generates protein polysaccharide by-products and secretes them to the extracellular, the fermentation broth becomes more and more viscous, the oxygen mass transfer is affected, the utilization rate of dissolved oxygen is lower and lower, the growth rate of L-histidine is slower, the fermentation period is prolonged, and the sugar acid conversion rate is low.

[0006] Document 2: Role of poly-gamma-glutamic acid molecular structure change in reducing viscosity of fermentation broth by potassium chloride, author Li Lingfu et al.

[0007] Document 2 discloses that the addition of KCl changes the molecular structure of PGA, and the change in molecular structure directly leads to a decrease in the viscosity of the PGA aqueous solution, thereby causing a decrease in the viscosity of the PGA fermentation broth. The molecular structure change when KCl reduces the viscosity of the fermentation broth involves molecular mass, stereoconfiguration and conformation, while the molecular structure change caused by other methods of reducing the viscosity of the PGA fermentation broth is often one-sided. For example, when B. subtilis (chungkookjang) is used to ferment PGA, the viscosity of the fermentation broth can be reduced by adding NaCl. The reason why the addition of NaCl causes a decrease in the viscosity of the fermentation broth is that NaCl leads to a decrease in the molecular mass of PGA. When B. subtilis CGMCC2108 is used to ferment PGA, the viscosity of the fermentation broth can be reduced by adding CaCl2 to the culture medium, and the reason why CaCl2 causes a decrease in the viscosity of the fermentation broth is that CaCl2 changes the conformation of PGA. The synergistic effect of the multi-faceted change in the molecular structure leading to a decrease in the viscosity of the PGA fermentation broth provides a new idea and basis for developing viscosity reduction strategies.

[0008] Documents 1 and 2 disclose the viscosity change trend of the fermentation broth in different fermentation environments during fermentation. According to the data published in the Journal of Fermentation Engineering in 2021, the viscosity of C. glutamicum during the middle stage of fermentation can reach 200-300 mpa.s. The reasons affecting the viscosity of the fermentation broth include: (1) a large amount of polysaccharides and proteins are secreted during the metabolic process, leading to an increase in viscosity; (2) culture medium composition: the type of carbon source directly affects the viscosity, and when glucose is used as the carbon source, the viscosity of the fermentation broth is usually lower than that of starch-based carbon sources (such as corn starch), which can increase the viscosity by more than 50% due to incomplete hydrolysis; (3) fermentation parameters, for example, if the fermentation temperature reaches the autolysis temperature of the bacterial cells, a large amount of nucleic acids and other substances will be released, causing the viscosity to rapidly increase; if the pH value is out of control, bacterial cell flocculation is likely to occur, and the viscosity will fluctuate significantly. When the dissolved oxygen is lower than 20% saturation, the bacteria switch to anaerobic metabolism, producing more extracellular polymers, and the viscosity can double.

[0009] From the above disclosed content and prior art, it can be known that the fermentation parameters and fermentation environment are relatively easy to control, and the cost of using glucose as the fermentation carbon source is higher than that of corn starch, but the concentration of the fermentation broth is relatively lower, which is more conducive to reducing the viscosity of the fermentation broth without considering the cost. In addition, substances such as potassium chloride and calcium chloride can be added to regulate the fermentation process, so that the amount of extracellular polysaccharides produced during fermentation is reduced, thereby improving the viscosity.

[0010] Document 3: Effect of calcium gluconate on L-histidine fermentation and condition optimization, author Zhao Jianyun.

[0011] Document 3 can be seen by measuring the change of glucose acid kinase specific activity that 20 g of calcium gluconate per liter of culture medium can induce the synthesis of glucose acid kinase; directly synthesize 6-P-gluconic acid into the HMP pathway; thereby increasing the flux of the HMP pathway. Since the proportion of HMP pathway flux in glucose catabolism is less than 20, increasing the flux of the HMP pathway has a great impact on histidine fermentation; on the one hand, it promotes the synthesis of the cell to improve the DCW, and on the other hand, it provides more precursor substances for the synthesis of histidine, and the acid production rate and conversion rate of histidine are greatly improved.

[0012] Document 4: Influence of sodium citrate on the metabolic flow distribution of L-histidine fermentation, authors Zhu Wenze et al. Document 4 discloses that adding sodium citrate at the beginning of fermentation can change the metabolic flow distribution of 6-phosphogluconate, pyruvate and acetyl coenzyme A, the key nodes of the L-histidine biosynthesis pathway, maintain the metabolic flow balance between the glycolysis pathway, the tricarboxylic acid cycle and the HMP, and is beneficial to improve the metabolic flow of the L-histidine biosynthesis pathway. Finally, the metabolic flow to histidine is increased by 7.86%.

[0013] Document 5: Xylose and glucose co-fermentation for producing L-histidine, authors Wu Huyun et al.

[0014] Document 5 discloses that the optimal concentration of the inducer xylose is 10 g / L by means of shake flask fermentation; the optimal induction time is 8 h of fermentation by means of 5 L fermenter fermentation; since xylose will be consumed during fermentation, the xylose concentration in the fermentation broth is maintained by adding xylose multiple times or knocking out the xylA gene to block the xylose metabolic pathway to stabilize the induction conditions. The results show that xylose as an inducer and carbon source is more conducive to the fermentation production of L-histidine. Therefore, the fermentation process for co-fermentation of xylose and glucose to produce L-histidine is explored. The main control points of the process are as follows: adding 10 g / L of xylose after 8 h of fermentation, and adding sugar solution with a mass ratio of xylose to glucose of 1:5 during the fermentation process. The final yield of L-histidine can reach 56.5 g / L, which is twice that of pure glucose fermentation.

[0015] References 3, 4, and 5 respectively introduced inducing agents calcium gluconate, sodium citrate, and xylose during the fermentation production of histidine to increase the flux of the HMP pathway, ultimately leading to increased histidine yield. Calcium gluconate and sodium citrate dissolve and ionize upon addition to the fermentation broth, generating large amounts of calcium and sodium ions in the solution, with calcium ion concentrations reaching 20 g / L and sodium ion concentrations reaching 2 g / L. Excessive metal ion content can affect the normal metabolism of the strain. For example, appropriate amounts of calcium ions can promote strain growth and metabolism, maintain the stability of yeast cell membranes, and enhance their activity, thereby accelerating fermentation. Generally, a calcium ion content of 30-80 mg / L in water is favorable for fermentation. If the calcium ion content is too low, strain growth may be inhibited, leading to slow fermentation; while excessively high content may affect the balance of other microorganisms, producing undesirable flavor substances and increasing impurities.

[0016] Secondly, although xylose does not affect the total ion concentration in the fermentation broth and does not produce adverse fermentation reactions, it is metabolized. After being consumed by the bacterial strain as a carbon source, it is necessary to replenish an appropriate amount of xylose in a timely manner. Alternatively, a gene knockout method could be used to block the xylose metabolic pathway, thereby maintaining a suitable induction concentration of xylose in the fermentation broth. However, gene knockout methods would significantly increase costs and technical difficulty.

[0017] Reference 6: CN112481325A A method for producing histidine using Serratia marcescens with added glucose.

[0018] Reference 6 discloses that the acid production mechanisms and tolerance to stimulating factors vary considerably among different strains, but this information is not particularly relevant. Existing technologies have conducted extensive research on the acid production mechanisms 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. However, sodium malate has no significant stimulating effect on Serratia marcescens ATCC31026. On the other hand, the ability of Serratia marcescens to produce histidine is significantly improved by adding calcium acetate and malonic acid, indicating that inducers are not universal and need to be tested and selected based on specific strains.

[0019] Reference 7: Qualitative and quantitative determination of L-histidine in fermentation broth, by He Junfeng.

[0020] Reference 7 discloses a method for detecting L-histidine in fermentation broth. Pauly colorimetry can preliminarily qualitatively identify histidine in the fermentation broth, but imidazole derivatives that may be present in the broth can interfere with this qualitative analysis. However, combining Pauly colorimetry with ninhydrin colorimetry can accurately qualitatively identify histidine in the fermentation broth. The addition of Pauly reagent B will cause turbidity in the fermentation broth supernatant, and ammonium ions have a certain influence on Pauly colorimetry; therefore, the Pauly colorimetric method is not suitable for the quantitative determination of histidine in fermentation broth. For the quantitative determination of histidine in fermentation broth, paper chromatography followed by elution and then colorimetric analysis is used. Summary of the Invention

[0021] The purpose of this invention is to provide a method for the purification and production of histidine. The technical problems to be solved by this invention are: (1) reducing the viscosity of the fermentation broth; (2) using a non-ionic inducer that does not affect the ion concentration of the fermentation broth after being added to it, and is not metabolized by the strain. That is, the inducer does not need to be replenished at any time during the fermentation process. The addition of the inducer can increase the final yield of histidine.

[0022] To achieve the above objectives, one embodiment of the present invention provides a method for purifying and producing histidine, comprising the following steps: Step S1: Prepare Corynebacterium glutamicum seed culture. Inoculate the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium for fermentation. The fermentation medium includes 30g / L~60g / L glucose, 30g / L~60g / L corn flour, 10g / L~15g / L ammonium sulfate, 2g / L~4g / L potassium dihydrogen phosphate, 0.1g / L~0.3g / L magnesium sulfate, and 10g / L~15g / L calcium carbonate. The fermentation temperature is 28℃~35℃, the fermentation pH is maintained at 7.0~7.5, and the dissolved oxygen concentration is 25%~35%. After 6~10h of fermentation, add 1g / L~3g / L geniposide and 20mg / L~50mg / L zinc sulfate as inducers. Monitor the glucose concentration during fermentation. Fermentation is completed after 45h~50h, and the fermentation broth is obtained. The Corynebacterium glutamicum BNCC186030 used in this invention was purchased from Beina Biotechnology - Henan Provincial Industrial Microbial Strains Engineering Technology Research Center.

[0023] Step S2: Filter the fermentation broth, adjust the pH value to the isoelectric point of histidine, then concentrate until crystals precipitate, wash and dry to obtain crude histidine. Step S3: Purification of crude histidine: Pure water was added to crude histidine and heated to dissolve it. After dissolution, activated carbon was added for adsorption and decolorization. After decolorization, acidic ion exchange resin was used for adsorption, and ammonia water was used for elution. The eluent was concentrated and crystallized, and then dried after cooling to obtain refined histidine.

[0024] In a preferred embodiment of the present invention, the preparation method of Corynebacterium glutamicum seed culture in step S1 is as follows: a strain of Corynebacterium glutamicum is streaked onto a slant culture medium for activation, and incubated at a constant temperature of 28-35°C for 24-30 hours; the activated strain is inoculated into a seed culture medium and incubated on a shaker at a constant temperature for 15-20 hours; then, the strain in the seed culture medium is inoculated into a fermentation culture medium at an inoculation rate of 5%-10%, and incubated at a constant temperature with shaking for 48-72 hours to obtain the Corynebacterium glutamicum seed culture.

[0025] Preferably, in step S1, the inoculum amount is 1% to 2%, and the pH value is maintained at 7.0 to 7.5 by adding ammonia water.

[0026] In a preferred embodiment of the present invention, in step S2, the pH value is adjusted to 7.4 to 7.6, and after concentration, the solution is washed several times with ammonia water with a pH value of 7.4 to 7.6.

[0027] In a preferred embodiment of the present invention, the dissolution temperature in step S3 is 30℃~50℃, the amount of activated carbon added is 2%~5%, the flow rate during ammonia elution is controlled at 1L / min~1.5L / min, and the drying temperature is 90℃~100℃.

[0028] In summary, the present invention has the following advantages: This invention optimizes the fermentation process of histidine by adding geniposide in the middle and late stages of fermentation to induce the HMP pathway flow, thereby increasing histidine production. The absence of geniposide in the early stages is intended to maintain a balance in the metabolic flow of glycolysis, the tricarboxylic acid cycle, and the HMP and EMP pathways during the fermentation period, ensuring the necessary environment for rapid bacterial growth.

[0029] This invention also purifies crude histidine using acidic ion exchange resin and concentrates and refines it through crystallization to obtain high-purity histidine. This invention increases the corn flour content in the fermentation medium and appropriately reduces the amount of glucose, thereby lowering production costs to some extent.

[0030] The addition of geniposide in this invention also plays a certain regulatory role in the middle and late stages of fermentation, resulting in a decrease in the content of extracellular polysaccharides in the fermentation broth and a decrease in the viscosity of the fermentation broth. Detailed Implementation

[0031] This invention provides a method for purifying and producing histidine, comprising the following steps: Step S1: Prepare Corynebacterium glutamicum seed culture. Inoculate the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium for fermentation. The fermentation medium includes glucose 30 g / L~60 g / L, corn flour 30 g / L~60 g / L, ammonium sulfate 10 g / L~15 g / L, potassium dihydrogen phosphate 2 g / L~4 g / L, magnesium sulfate 0.1 g / L~0.3 g / L, and calcium carbonate 10 g / L~15 g / L. The fermentation temperature is 28℃~35℃, the fermentation pH is maintained at 7.0~7.5, and the dissolved oxygen concentration is 25%~35%. After 6~10 h of fermentation, add the inducing agent geniposide 1 g / L~3 g / L and zinc sulfate 20 mg / L~50 mg / L. Monitor the glucose concentration during fermentation. Fermentation is completed after 45 h~50 h, yielding the fermentation broth. Step S2: Filter the fermentation broth, adjust the pH value to the isoelectric point of histidine, then concentrate until crystals precipitate, wash and dry to obtain crude histidine. Step S3: Purification of crude histidine: Pure water was added to crude histidine and heated to dissolve it. After dissolution, activated carbon was added for adsorption and decolorization. After decolorization, acidic ion exchange resin was used for adsorption, and ammonia water was used for elution. The eluent was concentrated and crystallized, and then dried after cooling to obtain refined histidine.

[0032] The present invention also discloses the preparation method of Corynebacterium glutamicum seed liquid in step S1 as follows: pick Corynebacterium glutamicum strain and streak it onto a slant culture medium for activation, and incubate it at a constant temperature of 28~35℃ for 24h~30h; inoculate the activated strain into the seed culture medium, place it on a shaker and incubate it at a constant temperature of 15h~20h, and then inoculate the strain in the seed culture medium into the fermentation culture medium at an inoculation rate of 5%~10%, and incubate it at a constant temperature of 48h~72h to obtain Corynebacterium glutamicum seed liquid.

[0033] Example A1: Preparation method of Corynebacterium glutamicum seed culture A strain of Corynebacterium glutamicum was streaked onto a slant culture medium for activation and incubated at 35°C for 24 hours. The activated strain was then inoculated into a seed culture medium and incubated on a shaker for 15 hours. Finally, the strain from the seed culture medium was inoculated into the fermentation medium at a 5% inoculation rate and incubated at a shaker for 72 hours to obtain the Corynebacterium glutamicum seed culture.

[0034] Example A2: Preparation method of Corynebacterium glutamicum seed culture A strain of Corynebacterium glutamicum was streaked onto a slant culture medium for activation and incubated at 28°C for 30 hours. The activated strain was then inoculated into a seed culture medium and incubated on a shaker for 20 hours. Finally, the strain from the seed culture medium was inoculated into the fermentation medium at a 10% inoculation rate and incubated at a shaker for 48 hours to obtain the Corynebacterium glutamicum seed culture.

[0035] Example 11: Preparation of crude histidine Step S1: Prepare Corynebacterium glutamicum seed culture. Inoculate the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium at an inoculation rate of 1.5%. The fermentation medium consists of 50 g / L glucose, 50 g / L corn flour, 12 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 0.18 g / L magnesium sulfate, and 12 g / L calcium carbonate. The fermentation temperature is 32℃, and the pH is maintained at 7.0-7.5 by adding ammonia water. The dissolved oxygen concentration is 30%. After 8 hours of fermentation, add 2 g / L geniposide and 35 mg / L zinc sulfate as inducers. Monitor the glucose concentration during fermentation. Fermentation is completed after 45-50 hours, yielding the fermentation broth. Step S2: Filter the fermentation broth, adjust the pH to 7.4-7.6 to the isoelectric point of histidine, then concentrate until crystals precipitate, wash twice with ammonia water at pH 7.4-7.6, and then dry to obtain crude histidine.

[0036] Example 12: Preparation of crude histidine Step S1: Prepare Corynebacterium glutamicum seed culture. Inoculate the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium at an inoculation rate of 1%. The fermentation medium consists of 30 g / L glucose, 60 g / L corn flour, 10 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, and 10 g / L calcium carbonate. The fermentation temperature is 28℃, and the pH is maintained at 7.0-7.5 by adding ammonia water. The dissolved oxygen concentration is 25%. After 6 hours of fermentation, add 1 g / L geniposide and 20 mg / L zinc sulfate as inducers. Monitor the glucose concentration during fermentation. Fermentation is completed after 45 hours, yielding the fermentation broth. Step S2: Filter the fermentation broth, adjust the pH to 7.4-7.6 to the isoelectric point of histidine, then concentrate until crystals precipitate, wash several times with ammonia water at pH 7.4-7.6, and then dry to obtain crude histidine.

[0037] Example 13: Preparation of crude histidine Step S1: Prepare Corynebacterium glutamicum seed culture. Inoculate the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium at an inoculation rate of 2%. The fermentation medium consists of 60 g / L glucose, 30 g / L corn flour, 15 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate, and 15 g / L calcium carbonate. The fermentation temperature is 35℃, and the pH is maintained at 7.0-7.5 by adding ammonia water. The dissolved oxygen concentration is 35%. After 10 hours of fermentation, add 3 g / L geniposide and 50 mg / L zinc sulfate as inducers. Monitor the glucose concentration during fermentation. Fermentation is completed after 50 hours, yielding the fermentation broth. Step S2: Filter the fermentation broth, adjust the pH to 7.4-7.6 to the isoelectric point of histidine, then concentrate until crystals precipitate, wash several times with ammonia water at pH 7.4-7.6, and then dry to obtain crude histidine.

[0038] Example 21: Purification of crude histidine Step S3: Add pure water to crude histidine and heat to 50°C to dissolve it; after dissolution, add activated carbon for adsorption and decolorization, with the amount of activated carbon added being 3%; after decolorization, use acidic ion exchange resin for adsorption and use ammonia water for elution, with the flow rate of ammonia water controlled at 1.2 L / min during elution; concentrate and crystallize the eluent, and after cooling, dry it at 100°C to obtain refined histidine.

[0039] Example 22: Purification of crude histidine Step S3: Add pure water to crude histidine and heat to 40°C to dissolve it; after dissolution, add activated carbon for adsorption and decolorization, with the amount of activated carbon added being 5%; after decolorization, use acidic ion exchange resin for adsorption and use ammonia water for elution, with the flow rate of ammonia water controlled at 1.5L / min during elution; concentrate and crystallize the eluent, and after cooling, dry it at 90°C to obtain refined histidine.

[0040] The crude histidine used in Examples 21 and 22 of this invention is derived from the crude histidine prepared by the method in Example 11 of this invention.

[0041] Experimental Example 1: The histidine concentrate prepared by the methods in Examples 21 and 22 of this invention was subjected to quality testing according to the standard test items listed in the Chinese Pharmacopoeia. The test results are shown below:

[0042] As can be seen from the above test results, the histidine concentrate obtained in Examples 21 and 22 of this invention meets the Chinese Pharmacopoeia standard, and all test indicators are qualified.

[0043] Experimental Example 2: Effect of Fermentation Inducer on Histidine Yield Experimental Example 21: Production Method of High-Quality Histidine Step S1: Prepare Corynebacterium glutamicum seed culture. Inoculate the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium at an inoculation rate of 1.5%. The fermentation medium consists of 50 g / L glucose, 50 g / L corn flour, 12 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 0.18 g / L magnesium sulfate, and 12 g / L calcium carbonate. The fermentation temperature is 32℃, and the pH is maintained at 7.0–7.5 by adding ammonia water. The dissolved oxygen concentration is 30%. After 8 hours of fermentation, add 2 g / L geniposide and 35 mg / L zinc sulfate as inducers. Monitor the glucose concentration during fermentation. Fermentation is completed after 45–50 hours, yielding the fermentation broth.

[0044] Experimental Example 22: Production Method of High-Quality Histidine Step S1: Prepare Corynebacterium glutamicum seed culture. Inoculate the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium at an inoculation rate of 1.5%. The fermentation medium consists of 50 g / L glucose, 50 g / L corn flour, 12 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 0.18 g / L magnesium sulfate, and 12 g / L calcium carbonate. The fermentation temperature is 32℃, and the pH is maintained at 7.0–7.5 by adding ammonia water. The dissolved oxygen concentration is 30%. The glucose concentration is monitored during fermentation. Fermentation is completed after 45–50 hours, yielding the fermentation broth.

[0045] No geniposide and zinc sulfate were added in Experiment 22.

[0046] Experimental Example 23: Production Method of High-Quality Histidine Step S1: Prepare Corynebacterium glutamicum seed culture. Inoculate the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium at an inoculation rate of 1.5%. The fermentation medium consists of 50 g / L glucose, 50 g / L corn flour, 12 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 0.18 g / L magnesium sulfate, and 12 g / L calcium carbonate. The fermentation temperature is 32℃, and the pH is maintained at 7.0–7.5 by adding ammonia water. The dissolved oxygen concentration is 30%. After 8 hours of fermentation, add 2 g / L of geniposide as an inducer. Monitor the glucose concentration during fermentation. Fermentation is completed after 45–50 hours, yielding the fermentation broth.

[0047] Zinc sulfate was not added in Experiment 23.

[0048] Experimental Example 24: Production Method of High-Quality Histidine Step S1: Prepare Corynebacterium glutamicum seed culture. Inoculate the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium at an inoculation rate of 1.5%. The fermentation medium consists of 50 g / L glucose, 50 g / L corn flour, 12 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 0.18 g / L magnesium sulfate, and 12 g / L calcium carbonate. The fermentation temperature is 32℃, and the pH is maintained at 7.0–7.5 by adding ammonia water. The dissolved oxygen concentration is 30%. At the beginning of fermentation, 2 g / L geniposide and 35 mg / L zinc sulfate are added as inducers. The glucose concentration is monitored during fermentation. Fermentation is completed after 45–50 hours, yielding the fermentation broth.

[0049] In Experiment 24, geniposide and zinc sulfate were added at the beginning of fermentation.

[0050] The fermentation state of histidine in Experiments 21 to 24 was detected, and the viscosity of the fermentation broth and the concentration of histidine were measured after fermentation. The results are shown below.

[0051]

[0052] This invention uses *Corynebacterium glutamicum*, whose fermentation broth viscosity is lower than that of *Serratia marcescens*, indicating that the viscosity of the fermentation broth produced by different strains varies. Furthermore, there are many histidine-fermenting strains, and different strains have different fermentation production capacities. Therefore, comparative experiments require the use of the same strain to avoid experimental errors caused by differences in strain genotypes.

[0053] A comparison of the experimental data above shows that the timing of adding geniposide and zinc sulfate does not significantly affect the final viscosity of the fermentation broth. A comparison between Experiment 22 and Experiment 23 shows that the viscosity of the fermentation broth in Experiment 23, which only added geniposide, is not much different from that in Experiment 22. However, after adding zinc sulfate to Experiment 23 (Experiment 21), the viscosity of the fermentation broth decreased significantly. This indicates that geniposide and zinc sulfate need to be added simultaneously to significantly reduce the metabolic level of extracellular polysaccharides, thereby inhibiting the expression of extracellular polysaccharides and reducing the flow rate of extracellular polysaccharide metabolic pathways during fermentation. Therefore, the final concentration of the fermentation broth in Experiment 21 is relatively lower.

[0054] Regarding the fermentation efficiency of histidine, Experiments 21 and 23 showed the highest histidine concentrations. However, zinc sulfate was not added in Experiment 23, indicating that the zinc sulfate content had no significant or substantial effect on the fermentation efficiency of histidine. Comparing Experiments 22 and 23, it was shown that the addition of geniposide significantly increased the fermentation concentration of histidine. Comparing Experiments 21 and 24, it can be seen that different addition times affected the fermentation efficiency of histidine. Since the addition of geniposide can affect the distribution of metabolic flux and induce the fermentation strain to shift towards the HMP pathway, and the strain is in a rapid growth phase at the beginning of fermentation, the metabolic network at this time involves complex synchronous reactions of multiple systems. If the induction of HMP pathway flux is enhanced at this time, the synthesis of other products may be affected. However, these products may be necessary elements for the growth and division of the fermenting bacteria. Therefore, adding geniposide at the beginning of fermentation can easily cause metabolic network imbalance, leading to instability or disharmony in the microbial community, resulting in a decrease in subsequent fermentation efficiency. When the inducer geniposide is added in the middle of fermentation, the entire fermentation system is relatively stable, the number of bacterial strains is large, and the elements required by each multi-enzyme reaction system are basically met for fermentation. At this time, the addition of geniposide to induce the fermenting bacteria to shift towards the HMP pathway will not damage the stability of the entire bacterial community and is conducive to the accumulation of the product histidine, thus significantly increasing the histidine concentration.

Claims

1. A method for the production of refined histidine, characterized in that, The method comprises the following steps: Step S1, preparing a seed liquid of corynebacterium glutamicum, inoculating the seed liquid of corynebacterium glutamicum into a fermentation tank containing a fermentation medium for fermentation; the fermentation medium comprises 30-60 g / L of glucose, 30-60 g / L of corn flour, 10-15 g / L of ammonium sulfate, 2-4 g / L of potassium dihydrogen phosphate, 0.1-0.3 g / L of magnesium sulfate, and 10-15 g / L of calcium carbonate; the fermentation temperature is 28-35 DEG C, the pH value of the fermentation is maintained at 7.0-7.5, and the dissolved oxygen concentration is 25%-35%; 1-3 g / L of an inducer geniposide and 20-50 mg / L of zinc sulfate are added after 6-10 hours of fermentation; the glucose concentration is detected during the fermentation, the fermentation is completed after 45-50 hours of fermentation, and a fermentation liquor is obtained; Step S2, filtering the fermentation liquor, adjusting the pH value to the isoelectric point of histidine, then concentrating to precipitate crystals, and drying after washing to obtain a crude histidine product; Step S3, refining the crude histidine product: adding pure water to the crude histidine product and dissolving by heating; after dissolving, adding activated carbon for adsorption and decolorization; after decolorization, using an acid ion exchange resin for adsorption, using ammonia water for elution, concentrating and crystallizing the eluate, and drying after cooling to obtain a refined histidine product.

2. The method for refined production of histidine according to claim 1, characterized in that: The preparation method of the seed liquid of corynebacterium glutamicum in step S1 is as follows: picking corynebacterium glutamicum strains and inoculating them on a slope medium for activation, and incubating at 28-35 DEG C for 24-30 hours; inoculating the activated strains into a seed culture medium, and incubating at a constant temperature on a shaker for 15-20 hours; then inoculating the strains in the seed culture medium into a fermentation medium at an inoculation amount of 5%-10%, and incubating at a constant temperature for 48-72 hours to obtain the seed liquid of corynebacterium glutamicum.

3. The method for refined production of histidine according to claim 1, characterized in that: The inoculation amount in step S1 is 1%-2%, and the pH value is maintained at 7.0-7.5 by adding ammonia water.

4. The method for refined production of histidine according to claim 1, characterized in that: In step S2, the pH value is adjusted to 7.4-7.6, and the ammonia water with a pH value of 7.4-7.6 is used for washing several times after concentration.

5. The method for refined production of histidine according to claim 1, characterized in that: In step S3, the dissolving temperature is 30-50 DEG C, the addition amount of activated carbon is 2%-5%, the flow rate of ammonia water during elution is controlled at 1-1.5 L / min, and the drying temperature is 90-100 DEG C.

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