Preparation method of high-purity valine
By optimizing the microbial fermentation method and utilizing Bacillus and Escherichia coli fermentation processes, combined with strict control of fermentation parameters and purification steps, the problem of low purity of valine in microbial fermentation was solved, and the preparation of high-efficiency and high-purity valine was achieved.
Patent Information
- Application Number
- CN202411254442.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-09
- Publication Date
- 2025-01-07
AI Technical Summary
Existing methods for preparing valine using microbial fermentation produce low purity, while chemical synthesis methods are costly and complex, making it difficult to meet the demand for high purity.
Fermentation was carried out using two strains of Bacillus and Escherichia coli. The fermentation process was optimized, and the aeration rate and rotation speed during fermentation were controlled. Bacillus and Escherichia coli were added in batches, and the fermentation medium components and purification steps were strictly controlled, including membrane filtration, heating concentration, activated carbon decolorization, and ion exchange resin adsorption.
The purity of valine was significantly improved to 99.981%, while maintaining high yield, thus achieving efficient and economical preparation of high-purity valine.
Smart Images
Figure BDA0005033542550000071 
Figure BDA0005033542550000081 
Figure BDA0005033542550000101
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of valine, and in particular to a method for preparing high-purity valine. Background Art
[0002] Valine is an essential glycogenic amino acid for the human body. It can promote normal growth, repair tissues, regulate blood sugar, and provide the necessary energy. When the human body is engaged in intense physical activity, valine can provide muscles with extra energy to produce glucose to prevent muscle weakness. It can also help the liver remove excess nitrogen (potential toxins) and transport nitrogen needed by the body to various parts. Since the body cannot produce valine itself, it must be supplemented through dietary sources. Its natural food sources include grains, dairy products, shiitake mushrooms, mushrooms, peanuts, soy protein and meat. However, although most people can get enough from their diet, cases of valine deficiency are still common. In addition, valine can treat liver failure and damage to these organs caused by alcoholism and drug abuse, and can also speed up wound healing. Therefore, how to artificially prepare valine has attracted people's attention.
[0003] At present, the preparation methods of valine are divided into chemical synthesis and microbial fermentation. The chemical synthesis method has high production cost, complex reaction, many steps, and many by-products; the microbial fermentation method has low raw material cost, mild reaction conditions, and can also achieve large-scale production. It is a very economical production method and occupies a dominant position in the preparation of valine. As people's research on microbial fermentation becomes more and more in-depth, the purity of valine produced by it has also been correspondingly required to be higher. Summary of the invention
[0004] In order to obtain valine with higher purity by microbial fermentation, the present application provides a method for preparing high-purity valine.
[0005] The present application provides a method for preparing high-purity valine, comprising the following steps: culturing a starting strain, fermenting to produce acid, purifying and post-treating to finally obtain high-purity valine, wherein the starting strains are Bacillus and Escherichia coli, and the specific operation of fermenting to produce acid is: inoculating the Bacillus seed liquid obtained after culturing into a fermentation medium, fermenting for a period of time under an environment with an aeration volume of 1-1.2vvm and a rotation speed of 150-180r / min, then inoculating the Escherichia coli seed liquid obtained after culturing into the culture medium, adjusting the aeration volume to 0.2-0.3vvm and the rotation speed to 80-100r / min, and continuing to ferment to obtain valine fermentation liquid.
[0006] Specifically, the specific operation of the culture is: streak the Bacillus in the starting strain on the slant medium, culture at a constant temperature of 30°C for 24 hours, then wash the bacterial lawn on the slant medium with water, and inoculate it into the primary medium at a 1% inoculation amount. This application takes broth agar medium as an example, and culture at a speed of 120r / min and a temperature of 30°C until OD 610 =6.0-10.0, and obtain the first-level seed shake flask inoculation, and inoculate the first-level seed shake flask inoculation into the secondary culture medium at an inoculum of 2%, and culture at a speed of 120r / min and a temperature of 30°C until the OD 610 =8.0-12.0, to obtain Bacillus seed solution; streak the Escherichia coli in the starting strain onto the slant medium, culture at 30°C for 24h, then wash the bacterial lawn on the slant medium with water, and inoculate it into the primary medium at a 1% inoculation amount. This application takes LB medium as an example, and culture at a temperature of 30°C until OD 610 =6.0-10.0, and obtain the first-level seed shake flask inoculation, and inoculate the first-level seed shake flask inoculation into the secondary culture medium at an inoculum of 2%, and culture at a temperature of 30°C until OD 610 =8.0-12.0, to obtain Escherichia coli seed solution; the secondary culture medium in the specific embodiment of the present application includes ammonium sulfate, potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, yeast powder, iron sulfate heptahydrate, manganese sulfate monohydrate, biotin, vitamin B 1 , kanamycin and glucose. Those skilled in the art can replace the components and adjust the addition amount of each component according to actual conditions.
[0007] Preferably, in the fermentation and acid production, the Bacillus seed liquid obtained after the culture is inoculated into the fermentation medium, and fermented for a period of time in an environment with an aeration volume of 1 vvm and a rotation speed of 175 r / min, and then the Escherichia coli seed liquid obtained after the culture is inoculated into the culture medium, and the fermentation is continued in an environment with an aeration volume of 0.25 vvm and a rotation speed adjusted to 85 r / min to obtain a valine fermentation liquid.
[0008] By adopting the above technical scheme, the present application utilizes two strains with good valine production ability in the fermentation acid production step in the preparation of high-purity valine, optimizes the fermentation process of different strains in the main fermentation period, adjusts the ventilation volume and rotation speed during fermentation to control the dissolved oxygen content in the system, and adds Bacillus and Escherichia coli in batches, respectively. At this time, the purity of the high-purity valine obtained is 99.955-99.961%. The ventilation volume after adding Bacillus is further controlled to be 1vvm and the rotation speed is 175r / min, and the ventilation volume after adding Escherichia coli is 0.25vvm and the rotation speed is adjusted to 85r / min, thereby gradually improving the purity of the high-purity valine finally obtained to 99.981%.
[0009] In the fermentation and acid production, the inoculation amount of the Bacillus seed liquid is 8-12%, and the inoculation amount of the Escherichia coli seed liquid is 4-6%.
[0010] Preferably, the inoculation amount of the Bacillus seed solution is 10%, and the inoculation amount of the Escherichia coli seed solution is 5%.
[0011] By adopting the above technical scheme, the present application also strictly controls the inoculation amount of Bacillus seed liquid and Escherichia coli seed liquid in fermentation acid production. If the inoculation amount is lower than 8% and 4% respectively, the strain growth will be slow, the fermentation cycle will be extended, and the valine yield in the valine fermentation liquid will be too low; if the inoculation amount is higher than 12% and 6% respectively, the bacterial growth and reproduction in the fermentation system will be vigorous, which will reduce the dissolved oxygen level, consume more nutrients to meet its own growth needs, increase metabolic waste, and thus reduce the purity of high-purity valine. Experimental data have shown that when the inoculation amount of Bacillus seed liquid is 10% and the inoculation amount of Escherichia coli seed liquid is 5%, the valine yield in the valine fermentation liquid and the purity of the high-purity valine finally obtained are both high, the yield can reach 5.23mg / L, and the purity can reach 99.975%.
[0012] Preferably, the fermentation medium includes vitamin B 1 , Vitamin B 3 As well as glucose, the vitamin B 1 The added amount is 4-10mg / L, vitamin B 3 The addition amount of is 10-40mg / L, and the addition amount of glucose is 60-100g / L.
[0013] Preferably, the vitamin B in the fermentation medium 1 The added amount is 5mg / L, vitamin B 3 The addition amount is 30mg / L.
[0014] Preferably, the amount of glucose added to the fermentation medium is 90 g / L.
[0015] By adopting the above technical solution, the present invention strictly controls the components in the fermentation medium and strictly controls the content of vitamin B 1 , Vitamin B 3and the respective addition amounts of glucose, thereby further improving the valine yield in the valine fermentation broth and the purity of the high-purity valine finally obtained. In a specific embodiment of the present application, the fermentation medium also includes ammonium sulfate, potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, yeast powder, ferrous sulfate heptahydrate, manganese sulfate monohydrate, biotin and a defoaming agent. The specific amounts of the above substances and the components of the fermentation medium can be routinely adjusted by those skilled in the art according to actual conditions. The components and amounts of the present application are only for illustration.
[0016] The purification and post-treatment comprises the following steps: filtering the valine fermentation broth through a membrane, then heating and concentrating, adjusting the pH, heating, adding activated carbon to the valine fermentation broth, stirring and decolorizing, and immediately filtering to obtain a filtrate after completion, lowering the temperature of the filtrate to below 20°C, performing a primary crystallization, centrifuging, obtaining crystals and a liquid, adding an ion exchange resin to the liquid for adsorption, then performing analysis, salting out, filter pressing, eluting, and then lowering the temperature of the obtained eluate to below 20°C, performing a secondary crystallization, centrifuging, mixing the crystals obtained at this time with the crystals obtained after the primary crystallization and centrifugation, and vacuum drying to obtain high-purity valine.
[0017] Optionally, the dosage of activated carbon used for decolorization in the purification and post-treatment is 1-3wt%.
[0018] Preferably, the dosage of activated carbon used for decolorization in the purification and post-treatment is 2 wt %.
[0019] Optionally, the ion exchange resin used for adsorption in the purification and post-treatment is any one of acrylic resin, phenolic resin and styrene strong acid resin.
[0020] Preferably, the ion exchange resin used for adsorption in the purification and post-treatment is a styrene-type strong acid resin.
[0021] The ion exchange resin needs to be pretreated before use, specifically: wash the ion exchange resin with water until there is no visible impurity, filter, then rinse with 1 mol / L NaOH solution, the elution dosage is 10 times the volume of the ion exchange resin, the elution time is 30 minutes, wash with deionized water until neutral, filter, then rinse with 1 mol / L HCl solution, the elution dosage is 10 times the volume of the ion exchange resin, the elution time is 30 minutes, wash with deionized water until neutral, and filter.
[0022] Optionally, the amount of ion exchange resin added for adsorption in the purification and post-treatment is 0.5-0.8 wt %.
[0023] Preferably, the amount of ion exchange resin added for adsorption in the purification and post-treatment is 0.7 wt %.
[0024] By adopting the above technical scheme, the present application performs a series of purification treatments and post-treatments on the valine fermentation broth, and finally obtains high-purity valine that is decolorized, impurity-removed and collected for a secondary period. First, the present application strictly controls the dosage of activated carbon in the decolorization. Although an increase in the dosage of activated carbon is beneficial to the removal of impurity pigments in valine, if the dosage of activated carbon is too large, the adsorption loss will also increase accordingly, which will reduce the purity of the valine finally obtained. Therefore, the present application gradually controls the dosage of activated carbon, which can further improve the purity of high-purity valine.
[0025] Secondly, the present application strictly controls the type of ion exchange resin. Compared with acrylic resin and phenolic resin, although styrene strong acid resin has a lower exchange capacity, there is a multi-molecular electrostatic neutralization effect between it and valine. The present application utilizes this effect to greatly improve the adsorption rate of valine in the ion exchange resin.
[0026] Finally, the present application strictly controls the amount of ion exchange resin added. If the amount added is too low, although the equilibrium adsorption capacity shows an increasing trend, it is unable to effectively adsorb all the valine in the liquid. If the amount added is too high, although more valine molecules diffuse to the site to participate in the exchange reaction, thereby improving the equilibrium adsorption capacity of the ion exchange resin, too much ion exchange resin will have an adverse effect on salting out, filter pressing, and elution, and will also reduce the purity of the product. Therefore, the present application gradually controls the amount of ion exchange resin added, which can further improve the purity of high-purity valine.
[0027] In a second aspect, the present application provides a high-purity valine prepared by the above preparation method, wherein the purity of the high-purity valine is ≥99.955%.
[0028] In summary, this application has the following beneficial technical effects: In the fermentation and acid production step of the preparation method of the present application, two strains both having good valine production capabilities are utilized, the fermentation process of the main fermentation period of different strains is optimized, the ventilation volume and rotation speed during fermentation are adjusted to control the dissolved oxygen content in the system, and Bacillus and Escherichia coli are added in batches and separately, assisted by the interlocking of subsequent steps, and the synergistic effect is fully exerted. The preparation method has a complete, mature and efficient valine preparation effect, can well improve the purity of the high-purity valine finally obtained, while taking into account the valine yield in the valine fermentation broth, and not only efficiently but also more highly purified high-purity valine is obtained. DETAILED DESCRIPTION
[0029] Material Source Unless otherwise specified, the raw materials used in this application are all commercially available products, specifically: Bacillus was provided by China Pharmaceutical Microbiological Culture Collection Center with the collection number CPCC 101200; Escherichia coli was provided by China Pharmaceutical Microbiological Culture Collection Center with the collection number CPCC 101501; LB medium was purchased from Shanghai Shenqi Biotechnology Co., Ltd.; Biotin was purchased from Shaanxi Chenming Biotechnology Co., Ltd., CAS No. 58-85-5; Yeast powder was purchased from Angel Yeast Co., Ltd., brand FM902; Kanamycin was purchased from Shandong Zhucheng Biotechnology Co., Ltd., CAS No. 25389-94-0; Broth agar medium was purchased from Wuhan Adama New Energy Co., Ltd.; Activated carbon was purchased from Gongyi Jingchuang Water Purification Filler Factory, with a particle size of 200 mesh, 5% moisture, and ash content ≤7%; Acrylic resin, phenolic resin and styrene strong acid resin were purchased from Jiangsu Industrial Chromatography Separation Engineering Technology Research Center, with models D113, HD-1 and 001×7 respectively; The present application is further described in detail below with reference to examples and comparative examples.
[0030] Example 1.1 A method for preparing high-purity valine comprises the following steps: S1. Cultivation: The Bacillus in the starting strain was streaked onto a slant medium and cultured at 30°C for 24 h. The bacterial lawn on the slant medium was then washed with water and inoculated into 100 mL of broth agar medium at a 1% inoculum. The whole was then placed in a 500 mL Erlenmeyer flask and cultured at a speed of 120 r / min and a temperature of 30°C until the OD reached 0. 610 =6.0, and the first-level seed shake flask inoculation was obtained. The first-level seed shake flask inoculation was inoculated into the secondary culture medium at an inoculum of 2%, and cultured at a speed of 120 r / min and a temperature of 30°C until the OD 610 =8.0, to obtain Bacillus seed solution; the Escherichia coli in the starting strain was streaked and activated on the slant medium, and cultured at 30°C for 24 hours, then the bacterial lawn on the slant medium was washed with water, inoculated into LB medium at a 1% inoculum, and then placed in a 120 mL conical flask and cultured at 30°C until OD 610 =6.0-10.0, and obtain the first-level seed shake flask inoculation, and inoculate the first-level seed shake flask inoculation into the secondary culture medium at an inoculum of 2%, and culture at a temperature of 30°C until OD 610 =8.0-12.0, to obtain E. coli seed solution; S2. Fermentation and acid production: The Bacillus seed liquid obtained after the culture was inoculated into the fermentation medium at an inoculation rate of 8%, and ammonia water was first added to control the pH value in the system to 7.3±0.1, and then fermented for 12 hours at a temperature of 30°C, a ventilation rate of 1vvm, and a rotation speed of 180r / min. Then, the Escherichia coli seed liquid obtained after the culture was inoculated into the medium at an inoculation rate of 6%, and the pH value was continued to be controlled at 7.3±0.1. The fermentation was continued for 24 hours at a temperature of 30°C, a ventilation rate of 0.2vvm, and a rotation speed of 100r / min to obtain a valine fermentation liquid. The fermentation medium consisted of 8g / L (NH 4 ) 2 SO 4 , 1g / L KH 2 PO 4 , 1g / LK 2 HPO 4 、0.5g / L MgSO 4 7H 2 O, 2.5 g / L yeast powder, 10 mg / L FeSO 4 7H 2 O, 4mg / L MnSO 4 ·H 2 O, 0.02mg / L biotin, 4mg / L vitamin B 1 , 40mg / L vitamin B 3 , 50mg / L kanamycin, 60g / L glucose and 1mL / L defoaming agent; S3. Purification and post-treatment: The valine fermentation broth obtained in step S2 is subjected to membrane filtration to obtain a filtrate, which is then concentrated by heating to a concentration of 40 g / L, the pH is adjusted to 5.4, and the filtrate is heated to 50° C. Activated carbon is added to the valine fermentation broth at a dosage of 1 wt%, and the bleaching is stirred for 20 min. After the filtrate is completed, it is immediately filtered while hot to obtain a filtrate, the filtrate temperature is reduced to below 20° C., a primary crystallization is performed, and centrifugation is performed to obtain crystals and a liquid, and a pretreated acrylic acid type resin is added to the liquid at an addition amount of 0.8 wt% for adsorption, the adsorption temperature is 25° C., the rotation speed is 120 r / min, and the adsorption time is 0.5 h. After the adsorption is completed, the acrylic acid type resin is dynamically desorbed using an analytical solution (hydrochloric acid and ethanol in a volume ratio of 1:1.3), the desorbed liquid is collected, and sodium chloride is added to the analytical solution until it is saturated for salting out, followed by filter pressing and elution, and the temperature of the eluate obtained is then reduced to below 20° C., a secondary crystallization is performed, and centrifugation is performed. The crystals obtained at this time are mixed with the crystals obtained after the primary crystallization and centrifugation, and vacuum dried to obtain high-purity valine.
[0031] Example 1.2 A method for preparing high-purity valine comprises the following steps: S1. Cultivation: The Bacillus in the starting strain was streaked onto a slant medium and cultured at 30°C for 24 h. The bacterial lawn on the slant medium was then washed with water and inoculated into 100 mL of broth agar medium at a 1% inoculum. The whole was then placed in a 500 mL Erlenmeyer flask and cultured at a speed of 120 r / min and a temperature of 30°C until the OD reached 0. 610 =6.0, and the first-level seed shake flask inoculation was obtained. The first-level seed shake flask inoculation was inoculated into the secondary culture medium at an inoculum of 2%, and cultured at a speed of 120 r / min and a temperature of 30°C until the OD 610 =8.0, to obtain Bacillus seed solution; the Escherichia coli in the starting strain was streaked and activated on the slant medium, and cultured at 30°C for 24 hours, then the bacterial lawn on the slant medium was washed with water, inoculated into LB medium at a 1% inoculum, and then placed in a 120 mL conical flask and cultured at 30°C until OD 610 =6.0-10.0, and obtain the first-level seed shake flask inoculation, and inoculate the first-level seed shake flask inoculation into the secondary culture medium at an inoculum of 2%, and culture at a temperature of 30°C until OD 610 =8.0-12.0, to obtain E. coli seed solution; S2. Fermentation and acid production: The Bacillus seed liquid obtained after the culture was inoculated into the fermentation medium at an inoculation rate of 8%, and ammonia water was first added to control the pH value of the system to 7.3±0.1, and then fermented for 12 hours at a temperature of 30°C, a ventilation rate of 1.1vvm, and a rotation speed of 165r / min. Then, the Escherichia coli seed liquid obtained after the culture was inoculated into the medium at an inoculation rate of 6%, and the pH value was continued to be controlled at 7.3±0.1. The fermentation was continued for 24 hours at a temperature of 30°C, a ventilation rate of 0.25vvm, and a rotation speed of 90r / min to obtain a valine fermentation liquid. The fermentation medium was composed of 8g / L (NH 4 ) 2 SO 4 , 1g / L KH 2 PO 4 , 1g / LK 2 HPO 4 、0.5g / L MgSO 4 7H 2 O, 2.5 g / L yeast powder, 10 mg / L FeSO 4 7H 2 O, 4mg / L MnSO 4 ·H 2 O, 0.02mg / L biotin, 4mg / L vitamin B 1 , 40mg / L vitamin B 3, 50mg / L kanamycin, 60g / L glucose and 1mL / L defoaming agent; S3. Purification and post-treatment: The valine fermentation broth obtained in step S2 is subjected to membrane filtration to obtain a filtrate, which is then concentrated by heating to a concentration of 40 g / L, the pH is adjusted to 5.4, and the filtrate is heated to 50° C. Activated carbon is added to the valine fermentation broth at a dosage of 1 wt%, and the bleaching is stirred for 20 min. After the filtrate is completed, it is immediately filtered while hot to obtain a filtrate, the filtrate temperature is reduced to below 20° C., a primary crystallization is performed, and centrifugation is performed to obtain crystals and a liquid, and a pretreated acrylic acid type resin is added to the liquid at an addition amount of 0.8 wt% for adsorption, the adsorption temperature is 25° C., the rotation speed is 120 r / min, and the adsorption time is 0.5 h. After the adsorption is completed, the acrylic acid type resin is dynamically desorbed using an analytical solution (hydrochloric acid and ethanol in a volume ratio of 1:1.3), the desorbed liquid is collected, and sodium chloride is added to the analytical solution until it is saturated for salting out, followed by filter pressing and elution, and the temperature of the eluate obtained is then reduced to below 20° C., a secondary crystallization is performed, and centrifugation is performed. The crystals obtained at this time are mixed with the crystals obtained after the primary crystallization and centrifugation, and vacuum dried to obtain high-purity valine.
[0032] Example 1.3 A method for preparing high-purity valine comprises the following steps: S1. Cultivation: The Bacillus in the starting strain was streaked onto a slant medium and cultured at 30°C for 24 h. The bacterial lawn on the slant medium was then washed with water and inoculated into 100 mL of broth agar medium at a 1% inoculum. The whole was then placed in a 500 mL Erlenmeyer flask and cultured at a speed of 120 r / min and a temperature of 30°C until the OD reached 0. 610 =6.0, and the first-level seed shake flask inoculation was obtained. The first-level seed shake flask inoculation was inoculated into the secondary culture medium at an inoculum of 2%, and cultured at a speed of 120 r / min and a temperature of 30°C until the OD 610 =8.0, to obtain Bacillus seed solution; the Escherichia coli in the starting strain was streaked and activated on the slant medium, and cultured at 30°C for 24 hours, then the bacterial lawn on the slant medium was washed with water, inoculated into LB medium at a 1% inoculum, and then placed in a 120 mL conical flask and cultured at 30°C until OD 610 =6.0-10.0, and obtain the first-level seed shake flask inoculation, and inoculate the first-level seed shake flask inoculation into the secondary culture medium at an inoculum of 2%, and culture at a temperature of 30°C until OD 610 =8.0-12.0, to obtain E. coli seed solution; S2. Fermentation and acid production: The Bacillus seed liquid obtained after the culture was inoculated into the fermentation medium at an inoculation rate of 8%, and ammonia water was first added to control the pH value in the system to 7.3±0.1, and then fermented for 12 hours at a temperature of 30°C, a ventilation rate of 1.2vvm, and a rotation speed of 150r / min. Then, the Escherichia coli seed liquid obtained after the culture was inoculated into the medium at an inoculation rate of 6%, and the pH value was continued to be controlled at 7.3±0.1. The fermentation was continued for 24 hours at a temperature of 30°C, a ventilation rate of 0.3vvm, and a rotation speed of 80r / min to obtain a valine fermentation liquid. The fermentation medium consisted of 8g / L (NH 4 ) 2 SO 4 , 1g / L KH 2 PO 4 , 1g / LK 2 HPO 4 、0.5g / L MgSO 4 7H 2 O, 2.5 g / L yeast powder, 10 mg / L FeSO 4 7H 2 O, 4mg / L MnSO 4 ·H 2 O, 0.02mg / L biotin, 4mg / L vitamin B 1 , 40mg / L vitamin B 3 , 50mg / L kanamycin, 60g / L glucose and 1mL / L defoaming agent; S3. Purification and post-treatment: The valine fermentation broth obtained in step S2 is subjected to membrane filtration to obtain a filtrate, which is then concentrated by heating to a concentration of 40 g / L, the pH is adjusted to 5.4, and the filtrate is heated to 50° C. Activated carbon is added to the valine fermentation broth at a dosage of 1 wt%, and the bleaching is stirred for 20 min. After the filtrate is completed, it is immediately filtered while hot to obtain a filtrate, the filtrate temperature is reduced to below 20° C., a primary crystallization is performed, and centrifugation is performed to obtain crystals and a liquid, and a pretreated acrylic acid type resin is added to the liquid at an addition amount of 0.8 wt% for adsorption, the adsorption temperature is 25° C., the rotation speed is 120 r / min, and the adsorption time is 0.5 h. After the adsorption is completed, the acrylic acid type resin is dynamically desorbed using an analytical solution (hydrochloric acid and ethanol in a volume ratio of 1:1.3), the desorbed liquid is collected, and sodium chloride is added to the analytical solution until it is saturated for salting out, followed by filter pressing and elution, and the temperature of the eluate obtained is then reduced to below 20° C., a secondary crystallization is performed, and centrifugation is performed. The crystals obtained at this time are mixed with the crystals obtained after the primary crystallization and centrifugation, and vacuum dried to obtain high-purity valine.
[0033] Examples 2.1-2.5 A method for preparing high-purity valine, which is different from Example 1.1 in that, in step S2, the ventilation volume and rotation speed before adjustment and the ventilation volume and rotation speed after adjustment are different, as shown in Table 1.1, and the rest are the same as Example 1.1.
[0034] Table 1.1 Ventilation volume and speed of Examples 2.1-2.5 Examples 3.1-3.8 A method for preparing high-purity valine, which is different from Example 1.1 in that in step S2, the inoculation amount of Bacillus seed liquid and the inoculation amount of Escherichia coli seed liquid are different, see Table 1.2 for details, and the rest are the same as Example 1.1.
[0035] Table 1.2 Inoculation amount of seed solution of Examples 3.1-3.8 Bacillus seed solution inoculation amount % E. coli seed solution inoculation amount % Example 1.1 8 6 Example 3.1 10 6 Example 3.2 12 6 Example 3.3 8 5 Example 3.4 10 5 Example 3.5 12 5 Example 3.6 8 4 Example 3.7 10 4 Example 3.8 12 4 Examples 3.9-3.10 A method for preparing high-purity valine, which is different from Example 2.3 in that in step S2, the inoculation amount of Bacillus seed liquid and the inoculation amount of Escherichia coli seed liquid are different, see Table 1.3 for details, and the rest are the same as Example 2.3.
[0036] Table 1.3 Inoculation amount of seed solution of Examples 3.9-3.10 Bacillus seed solution inoculation amount % E. coli seed solution inoculation amount % Example 2.3 8 6 Example 3.9 10 5 Example 3.10 12 4 Examples 4.1-4.6 A method for preparing high-purity valine, which is different from Example 1.1 in that, in step S2, vitamin B in the fermentation medium 1 and vitamin B 3 The addition amount is different, see Table 1.4 for details, and the rest is the same as Example 1.1.
[0037] Table 1.4 Vitamin B of Examples 4.1-4.6 1 , Vitamin B 3 Amount of addition <![CDATA[Vitamin B 1 Dosage added mg / L]]> <![CDATA[Vitamin B 3 Addition amount mg / L]]> Example 1.1 4 40 Example 4.1 7 25 Example 4.2 10 10 Example 4.3 6 20 Example 4.4 8 35 Example 4.5 5 30 Example 4.6 9 15 Examples 5.1-5.4 A method for preparing high-purity valine, which is different from Example 1.1 in that, in step S2, the amount of glucose added to the fermentation medium is different, namely 70 g / L, 80 g / L, 90 g / L and 100 g / L, and the rest is the same as Example 1.1.
[0038] Examples 6.1-6.4 A method for preparing high-purity valine is different from Example 1.1 in that, in step S3, the dosage of activated carbon is 1.5wt%, 2wt%, 2.5wt% and 3wt%, respectively, and the rest is the same as Example 1.1.
[0039] Examples 7.1-7.2 A method for preparing high-purity valine, which is different from Example 1.1 in that, in step S3, the pretreated acrylic resin is replaced by the pretreated phenolic resin and the pretreated styrene strong acid resin, respectively, and the rest is the same as Example 1.1.
[0040] Examples 8.1-8.3 A method for preparing high-purity valine is different from Example 1.1 in that, in step S3, the added amounts of the pretreated acrylic resin are 0.7wt%, 0.6wt% and 0.5wt%, respectively, and the rest are the same as Example 1.1.
[0041] Comparative Example 1 The difference from Example 1.1 is that steps S1 and S2 are specifically as follows: S1. Cultivation: The Bacillus in the starting strain was streaked onto a slant medium and cultured at 30°C for 24 h. The bacterial lawn on the slant medium was then washed with water and inoculated into 100 mL of broth agar medium at a 1% inoculum. The whole was then placed in a 500 mL Erlenmeyer flask and cultured at a speed of 120 r / min and a temperature of 30°C until the OD reached 0. 610 =6.0, and the first-level seed shake flask inoculation was obtained. The first-level seed shake flask inoculation was inoculated into the secondary culture medium at an inoculum of 2%, and cultured at a speed of 120 r / min and a temperature of 30°C until the OD 610 =8.0, and obtain Bacillus seed solution; S2. Fermentation and acid production: The Bacillus seed liquid obtained after culture is inoculated into the fermentation medium at an inoculation rate of 8%. First, ammonia water is added to control the pH value in the system to 7.3±0.1. Then, the mixture is fermented for 36 hours at a temperature of 30°C, a ventilation volume of 1vvm, and a rotation speed of 180r / min to obtain valine fermentation liquid.
[0042] Comparative Example 2 The difference from Example 1.1 is that steps S1 and S2 are specifically as follows: S1. Cultivation: The E. coli in the starting strain was streaked onto a slant medium and cultured at 30°C for 24 h. The bacterial lawn on the slant medium was then washed with water and inoculated into LB medium at a 1% inoculum. The whole was then placed in a 120 mL Erlenmeyer flask and cultured at 30°C until the OD 610=6.0-10.0, and obtain the first-level seed shake flask inoculation, and inoculate the first-level seed shake flask inoculation into the secondary culture medium at an inoculum of 2%, and culture at a temperature of 30°C until OD 610 =8.0-12.0, to obtain E. coli seed solution; S2. Fermentation and acid production: The Escherichia coli seed liquid obtained after culture was inoculated into the culture medium at an inoculation rate of 6%. Ammonia water was first added to control the pH value in the system to 7.3±0.1. The mixture was fermented for 36 hours at a temperature of 30°C, a ventilation volume of 0.2vvm, and a rotation speed of 100r / min to obtain valine fermentation liquid.
[0043] Comparative Example 3 The difference from Example 1.1 is that step S2 is specifically as follows: S2. Fermentation and acid production: The Bacillus seed liquid obtained after culture is inoculated into the fermentation medium at an inoculation rate of 8%, and then the Escherichia coli seed liquid obtained after culture is inoculated into the medium at an inoculation rate of 6%. First, ammonia water is added to control the pH value in the system to 7.3±0.1, and ferment for 36 hours at a temperature of 30°C, a ventilation volume of 0.2vvm, and a rotation speed of 100r / min to obtain valine fermentation liquid.
[0044] Comparative Examples 4.1-4.2 The difference from Example 1.1 is that in step S2, the ventilation volume and rotation speed before adjustment are different from the ventilation volume and rotation speed after adjustment, as shown in Table 1.5. The rest is the same as Example 1.1.
[0045] Table 1.5 Ventilation volume and speed of comparative examples 4.1-4.2 Performance Testing 1. Use liquid chromatography to detect the purity of high-purity valine, and record the results in Table 2; 2. The valine yield in the valine fermentation broth was determined by HPLC with o-phthalaldehyde pre-column derivatization. The detection limit of valine by liquid chromatography was 1 mg / L. The results are recorded in Table 2. 3. The high-purity valine obtained in Example 1.1 was tested according to the detection method of AJ192. The test items, test standards and test results are shown in Table 3.
[0046] Table 2 Purity of high-purity valine and valine yield of valine fermentation broth Table 3 Test report of Example 1.1 Data Analysis: As can be seen from Table 2, the purity of the high-purity valine of Examples 1.1-1.3 can reach 99.955-99.961%, and the valine yield in the valine fermentation broth can reach 5.19-5.21 mg / L, which proves that the steps in the preparation method of the present application cooperate with each other and are closely connected. Therefore, the preparation method has a complete, mature and efficient valine preparation effect, and can well improve the purity of the high-purity valine finally obtained, while taking into account the valine yield in the valine fermentation broth, and high-purity valine is prepared not only efficiently but also with higher quality; Example 2.3 The high-purity valine in Examples 2.1-2.5 has the highest purity, which proves that the present application controls the content of dissolved oxygen in the system by adjusting the ventilation volume and rotation speed during fermentation, thereby significantly improving the purity of the high-purity valine finally obtained; The purity and yield of high-purity valine in Example 3.4 in Examples 3.1-3.8 are both high, and the values of high-purity valine purity and valine yield in Example 3.9 are also at a high level, proving that the present application strictly controls the inoculation amount of Bacillus seed liquid and Escherichia coli seed liquid in fermentation acid production, so that the valine yield in the valine fermentation broth and the purity of the high-purity valine finally obtained are both high and in the best balance; The purity and yield of high-purity valine in Example 4.5 in Examples 4.1-4.6 are both high, and the purity and yield of high-purity valine in Example 5.3 in Examples 5.1-5.4 are both high, which proves that the present application further improves the yield of valine in the valine fermentation broth and the purity of the high-purity valine finally obtained by strictly controlling the addition amount of vitamin B1, vitamin B3 and glucose; Example 6.2 The high-purity valine in Examples 6.1-6.4 has the highest purity, which proves that the present application can further improve the purity of high-purity valine by gradually controlling the dosage of activated carbon; The purity of the high-purity valine in Example 7.2 is higher than that in Example 1.1 and Example 7.1, which proves that the purity of the high-purity valine can be further improved by gradually controlling the amount of ion exchange resin added in the present application; Example 8.1 The high-purity valine in Examples 8.1-8.3 has the highest purity, proving that the present application greatly improves the adsorption rate of valine in the ion exchange resin by utilizing the multi-molecular electrostatic neutralization effect between the styrene-type strong acid resin and valine; The purity of the high-purity valine in Comparative Examples 1-4.2 is much lower than that in Example 1.1, which proves that the present application optimizes the fermentation process of the main fermentation period of different strains by utilizing two strains both having good valine production ability, and adjusts the ventilation volume and rotation speed during fermentation to control the dissolved oxygen content in the system, and adds Bacillus and Escherichia coli in batches, respectively, so as to gradually improve the purity of the high-purity valine finally obtained.
[0047] The embodiments of this specific implementation method are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore, all equivalent changes made based on the structure, shape, and principle of the present application should be included in the protection scope of the present application.
Claims
1. A method for preparing high-purity valine, comprising the following steps: The starting strain is cultured, fermented to produce acid, purified and post-treated to finally obtain high-purity valine, wherein the starting strain is Bacillus and Escherichia coli, and the specific operation of the fermentation to produce acid is: The Bacillus seed liquid obtained after the culture is inoculated into the fermentation medium, and fermented for a period of time under the environment of a ventilation volume of 1-1.2vvm and a rotation speed of 150-180r / min. Subsequently, the Escherichia coli seed liquid obtained after the culture is inoculated into the medium, the ventilation volume is adjusted to 0.2-0.3vvm, the rotation speed is adjusted to 80-100r / min, and the fermentation is continued to obtain valine fermentation liquid.
2. The method for preparing high-purity valine according to claim 1, characterized in that: In the fermentation and acid production, the Bacillus seed liquid obtained after the culture is inoculated into the fermentation medium, and fermented for a period of time under the environment of a ventilation volume of 1vvm and a rotation speed of 175r / min. Subsequently, the Escherichia coli seed liquid obtained after the culture is inoculated into the culture medium, and the fermentation is continued under the environment of a ventilation volume of 0.25vvm and a rotation speed adjusted to 85r / min to obtain the valine fermentation liquid.
3. A method for preparing high-purity valine according to claim 1 or 2, characterized in that: The inoculation amount of the Bacillus seed liquid is 10%, and the inoculation amount of the Escherichia coli seed liquid is 5%.
4. The method for preparing high-purity valine according to claim 1, characterized in that: The fermentation medium comprises vitamin B1, vitamin B3 and glucose, wherein the added amount of vitamin B1 is 4-10 mg / L, the added amount of vitamin B3 is 10-40 mg / L, and the added amount of glucose is 60-100 g / L.
5. The method for preparing high-purity valine according to claim 4, characterized in that: The amount of vitamin B1 added to the fermentation medium is 5 mg / L, and the amount of vitamin B3 added is 30 mg / L.
6. The method for preparing high-purity valine according to claim 4, characterized in that: The amount of glucose added to the fermentation medium is 90 g / L.
7. The method for preparing high-purity valine according to claim 1, characterized in that: The dosage of activated carbon used for decolorization in the purification and post-treatment is 2 wt %.
8. The method for preparing high-purity valine according to claim 1, characterized in that: The ion exchange resin used for adsorption in the purification and post-treatment is a styrene-type strong acid resin.
9. The method for preparing high-purity valine according to claim 1, characterized in that: The amount of ion exchange resin added for adsorption in the purification and post-treatment is 0.7 wt %.
10. A high-purity valine obtained by the preparation method according to any one of claims 1 to 9, characterized in that: The purity of the high-purity valine is ≥99.955%.