A fermentation method for reducing the content of heteroacids in L-valine fermentation broth

Abstract

The present invention belongs to the technical field of preparing L-valine by microbial fermentation, and specifically relates to a fermentation method for reducing the content of heteroacids in L-valine fermentation broth. The present invention inoculates Escherichia coli into a fermentation medium for fermentation culture, and the ammonia nitrogen content in the fermentation broth is ≤0.12% (w / v), the stirring direction is counterclockwise rotation, and a glucose solution with a pH of 6.0-7.0 is added; the ammonia nitrogen content in the fermentation broth is greater than 0.12% and less than 0.15% (w / v), the stirring direction is clockwise rotation, and a glucose solution with a pH of 9.0-9.2 is added; the ammonia nitrogen content in the fermentation broth is ≥0.15% (w / v), the stirring direction is clockwise rotation, and a glucose solution with a pH of 10.0-10.2 is added. By changing the stirring direction and adding different pH glucose solutions, the ammonia nitrogen content in the fermentation broth is effectively controlled, the amount of heteroacids produced by microorganisms is reduced, and the L-valine fermentation conversion rate is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of preparing L-valine by microbial fermentation, and particularly relates to a fermentation method for reducing the content of heteroacids in L-valine fermentation liquid. Background Art

[0002] The chemical name of L-valine is α-amino-3-methylbutyric acid, and its molecular formula is C 5 H 11 NO 2 It is odorless, bitter, white monoclinic crystal or crystalline powder. L-valine is a neutral amino acid, its aqueous solution is weakly acidic, it belongs to branched chain amino acid, is one of the essential amino acids for human body, and is widely used in medicine, food additives and animal feed industries.

[0003] At present, microbial fermentation is the preferred production method of L-valine. It is a method that utilizes the biological metabolism of microorganisms to directly ferment and synthesize L-valine with glucose as raw material. This method has the advantages of low raw material cost, mild reaction conditions and easy control, and low pollution. However, the ammonia nitrogen content in the fermentation broth often continues to increase during the fermentation production of L-valine. This is because acidic substances are produced during the metabolism of the bacteria, and the optimal growth pH value of the bacteria is neutral. Therefore, liquid ammonia or ammonia water is usually required to maintain the pH value at a neutral level during the fermentation process. However, the continuous introduction of liquid ammonia or ammonia water will increase the concentration of free ammonium ions in the fermentation broth, and it cannot be quickly utilized by microorganisms. As the fermentation cycle is extended, the ammonia nitrogen content becomes higher and higher, and can even be as high as 0.5% or more. Under normal circumstances, the optimal ammonia nitrogen content in the fermentation broth during the production of amino acid fermentation is within 0.2%. Too high ammonia nitrogen content will not only affect the growth and reproduction of microorganisms, change the metabolic pathways of microorganisms, but also affect the accumulation of products, causing microorganisms to produce organic acids or other miscellaneous acids such as amino acids. In addition, too high ammonia nitrogen content will also lead to difficulties in the extraction process, further affecting product quality, which is not conducive to the green development of the industry.

[0004] Therefore, it is necessary to propose a fermentation method that can reduce the content of impurities in the fermentation broth by effectively controlling the ammonia nitrogen content in the fermentation broth within a reasonable range during the L-valine fermentation process, thereby improving the L-valine conversion rate and reducing the production cost. Summary of the invention

[0005] The object of the present invention is to provide a fermentation method for reducing the content of heteroacids in L-valine fermentation broth, by effectively controlling the ammonia nitrogen content in the fermentation broth to 0.04%-0.2% (w / v), reducing the synthesis of other heteroacids such as organic acids and other amino acids, and improving the fermentation conversion rate of L-valine.

[0006] In order to solve the above technical problems, the following technical solutions are proposed:

[0007] The present invention provides a fermentation method for reducing the content of heteroacids in L-valine fermentation broth, comprising the following steps:

[0008] Inoculating Escherichia coli into a fermentation medium for fermentation culture, and starting to feed glucose solution when the glucose concentration in the fermentation liquid is ≤0.2% (w / v), and obtaining a fermentation liquid containing L-valine after the fermentation is completed;

[0009] During the fermentation culture, when the ammonia nitrogen content in the fermentation liquid is ≤0.12%, the stirring direction is counterclockwise and a glucose solution with a pH value of 6.0-7.0 is added;

[0010] When the ammonia nitrogen content in the fermentation liquid is greater than 0.12% and less than 0.15%, the stirring direction is clockwise and a glucose solution with a pH value of 9.0 to 9.2 is added;

[0011] When the ammonia nitrogen content in the fermentation liquid is ≥0.15%, the stirring direction is clockwise and a glucose solution with a pH value of 10.0-10.2 is added; when the ammonia nitrogen content in the fermentation liquid drops to greater than 0.12% and less than 0.15%, the stirring direction is clockwise and a glucose solution with a pH value of 9.0-9.2 is added.

[0012] Preferably, the fermentation temperature is 35-38° C., the rotation speed is 150-200 rpm, and the fermentation time is 28-30 h.

[0013] Preferably, the fermentation pressure is 0.03-0.05 MPa, and the ventilation rate is 0.05-0.2 m3 / min per cubic meter of fermentation liquid. 3 Air.

[0014] Preferably, the pH value during the fermentation culture process is 6.9-7.1.

[0015] Preferably, the mass concentration of the glucose solution is 60% to 65%; after the glucose solution is added, the glucose concentration in the fermentation broth is controlled to be 0.1% to 0.2% (w / v).

[0016] Preferably, the fermentation medium uses water as a solvent and includes the following components in concentrations: 3-5 g / L yeast powder, 2-3 g / L corn steep liquor, 1-1.2 g / L betaine, 10-15 g / L glucose, 0.2-0.5 g / L magnesium sulfate, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L ammonium citrate, 3-6 mg / L cobalt chloride and 20-25 mg / L nicotinamide.

[0017] Preferably, the inoculation method of the Escherichia coli includes seed liquid; the inoculation amount of the seed liquid is 13% to 15% of the volume of the fermentation medium.

[0018] Preferably, the OD of the seed solution is 600 The value is 10~12.

[0019] Preferably, the preparation method of the seed solution comprises: inoculating Escherichia coli into a seed culture medium for seed culture to obtain the Escherichia coli seed solution; the seed culture medium uses water as a solvent and also includes components with the following concentrations: yeast powder 8~10g / L, glucose 25~30g / L, magnesium sulfate 0.5~0.8g / L, dipotassium hydrogen phosphate 2~4g / L, potassium dihydrogen phosphate 2~4g / L and ammonium sulfate 6~8g / L.

[0020] Preferably, the temperature of the seed culture is 35-37° C., the pH value during the seed culture process is 6.9-7.0, and the pressure of the seed culture is 0.03-0.05 MPa.

[0021] Beneficial effects of the present invention:

[0022] In the production of valine fermentation, as the fermentation culture time increases, the ammonia nitrogen content in the fermentation broth will continue to increase. When the content is greater than 0.2%, it will not only affect the growth and reproduction of microorganisms and change the metabolic pathways of microorganisms, but also affect the accumulation of products, causing microorganisms to produce organic acids or other amino acids and other miscellaneous acids. The present invention provides a fermentation method for reducing the content of miscellaneous acids in L-valine fermentation broth. The present invention produces valine by effectively controlling the ammonia nitrogen content in the fermentation production, thereby reducing the content of miscellaneous acids in the L-valine fermentation broth:

[0023] (1) When the volume is small and the foam is small at the beginning of fermentation, the stirring direction is set to counterclockwise rotation to make the fermentation liquid rise, increasing the contact area between the fermentation liquid and the air. The excess free ammonium ions evaporate quickly and are discharged from the fermentation tank along with the air, preventing the ammonia nitrogen content in the fermentation liquid from increasing rapidly.

[0024] (2) With the extension of fermentation culture time, the rapid reproduction of microorganisms and the gradual increase in the volume of fermentation liquid caused the fermentation foam to increase gradually. The free ammonium ions were difficult to be discharged from the fermentation tank quickly. The ammonium ions in the fermentation liquid could not be quickly consumed and utilized by microorganisms, resulting in a rapid increase in the ammonia nitrogen content. The ammonia nitrogen content in the fermentation liquid was reduced by restoring the stirring direction to clockwise and reducing the frequency of liquid ammonia introduction by adding glucose solutions with different pH values.

[0025] By implementing the present invention, the content of ammonia nitrogen in the fermentation liquid is controlled within the optimal range suitable for the fermentation production of valine, the synthesis of organic acids or other miscellaneous acids such as amino acids produced by Escherichia coli is reduced, and the fermentation conversion rate of L-valine is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0027] Figure 1 The content of ammonia nitrogen in the fermentation broth in the fermentation culture in Example 1; Figure 1 The fermentation cycle in is the total time of fermentation culture;

[0028] Figure 2 The content of ammonia nitrogen in the fermentation broth in the fermentation culture in Comparative Example 6 is as follows: Figure 2 The fermentation cycle is the total time of fermentation culture. DETAILED DESCRIPTION

[0029] The present invention provides a fermentation method for reducing the content of heteroacids in L-valine fermentation broth, comprising the following steps:

[0030] Escherichia coli is inoculated into a fermentation medium for fermentation culture. When the glucose concentration in the fermentation liquid is ≤0.2% (w / v), glucose solution is added. After the fermentation is completed, a fermentation liquid containing L-valine is obtained.

[0031] The mass concentration of the glucose solution added in the present invention is preferably 60% to 65%, more preferably 60% to 63%; after adding the glucose solution, the glucose concentration in the fermentation broth is preferably controlled to be 0.1% to 0.2% (w / v). Glucose is a substrate for Escherichia coli to ferment L-valine, and the addition of glucose solution can promote the production of L-valine.

[0032] In the present invention, there is no particular limitation on the strain type of the Escherichia coli, and any strain type of the Escherichia coli conventionally used for fermentation to prepare L-valine in the art can be used.

[0033] In the present invention, Escherichia coli is inoculated into a fermentation medium for fermentation culture, and the ammonia nitrogen content of the obtained fermentation liquid is in a changing state.

[0034] During the fermentation process, liquid ammonia needs to be introduced to maintain the pH value. Continuous introduction will increase the concentration of free ammonium ions in the fermentation liquid, and it cannot be quickly utilized by microorganisms. As the fermentation culture time increases, the ammonia nitrogen content gradually increases. In the present invention, the fermentation culture is divided into three stages according to the ammonia nitrogen content in the fermentation liquid, and the fermentation culture includes a first fermentation stage, a second fermentation stage, and a third fermentation stage.

[0035] In the first fermentation stage of the present invention, the ammonia nitrogen content in the fermentation liquid is ≤0.12% (w / v), the stirring direction is counterclockwise rotation and a glucose solution with a pH value of 6.0-7.0 is added. The natural pH value of the glucose solution after preparation is 6.0-7.0 and no adjustment is required. The counterclockwise rotation in the present invention refers to the counterclockwise rotation direction of the fermentation stirring when viewed from the top of the fermentation tank, which will raise the fermentation liquid.

[0036] In the first fermentation stage, the volume of the fermentation culture is small and the foam is less at the beginning. The fermentation liquid is stirred in a counterclockwise direction, and the fermentation liquid is turned upward during the rotation, thereby increasing the contact area between the fermentation liquid and the air. Excess free ammonium ions evaporate quickly and are discharged from the fermentation tank along with the air, thereby preventing the ammonium ion content in the fermentation liquid from increasing rapidly.

[0037] In the second fermentation stage of the present invention, the ammonia nitrogen content in the fermentation liquid is greater than 0.12% and less than 0.15% (w / v), the stirring direction is clockwise rotation and a glucose solution with a pH value of 9.0-9.2 is added, and more preferably a glucose solution with a pH value of 9.1 is added. The glucose solution is preferably added after adjusting the pH value to 9.0-9.2. The pH regulator of the glucose solution preferably includes potassium hydroxide; the potassium hydroxide can provide nutrients for the production of Escherichia coli. The technical features of the glucose solution and the technical features of the glucose concentration control in the fermentation liquid have been discussed above and will not be repeated here. The clockwise direction described in the present invention refers to the clockwise rotation direction of the fermentation stirring when viewed from the top of the fermentation tank, which will press down the fermentation liquid.

[0038] In the third fermentation stage of the present invention, when the ammonia nitrogen content in the fermentation broth is ≥0.15% (w / v), the stirring direction is clockwise rotation and a glucose solution with a pH value of 10.0-10.2 is added, and more preferably a glucose solution with a pH value of 10.1 is added. The glucose solution is preferably added after adjusting the pH value to 10.0-10.2. The pH value regulator of the glucose solution preferably includes potassium hydroxide. The technical features of the glucose solution and the technical features of controlling the glucose concentration in the fermentation broth have been discussed above and will not be repeated here.

[0039] In the second fermentation stage and the third fermentation stage, with the extension of the fermentation cycle, the rapid reproduction of microorganisms and the gradual increase in the volume of the fermentation liquid, the fermentation foam gradually increases, and the free ammonium ions are difficult to quickly discharge from the fermentation tank. At the same time, when the stirring direction is counterclockwise, the amount of foam in the fermentation liquid will increase, which is easy to cause the liquid to overflow and cause bacterial contamination; and in order to control the fermentation pH value at 6.9~7.1 during the fermentation process, the frequency of liquid ammonia introduction is gradually increased, and the ammonium ions in the fermentation liquid cannot be quickly consumed and utilized by microorganisms. It is also difficult to reduce the ammonia nitrogen content in the fermentation liquid by raising the fermentation liquid, resulting in a rapid increase in the ammonia nitrogen content. At this time, the stirring direction is restored to clockwise to press down the fermentation liquid to prevent the fermentation liquid from overflowing seriously and causing bacterial contamination, and by adding glucose solutions with different pH values, the frequency of liquid ammonia introduction is reduced, thereby reducing the ammonia nitrogen content in the fermentation liquid, and controlling the ammonia nitrogen content in the fermentation liquid within the optimal range suitable for producing valine.

[0040] As the glucose solution with a pH value of 10.0-10.2 is added in the third fermentation stage, the ammonia nitrogen content in the fermentation liquid is in a decreasing state. When the ammonia nitrogen content in the fermentation liquid drops to more than 0.12% and less than 0.15% (w / v), the second fermentation stage begins again, and the glucose solution with a pH value of 9.0-9.2 is added. The fermentation culture is continued, and the second fermentation stage or the third fermentation stage is determined according to the ammonia nitrogen content in the fermentation liquid until the fermentation ends.

[0041] In the fermentation tank used in the present invention, the stirring device is a stirring blade, and the stirring blade has an inclination. Clockwise rotation will press down the fermentation liquid, and counterclockwise rotation will lift up the fermentation liquid.

[0042] In the present invention, each fermentation cycle of each fermentation culture is carried out according to the parameters of the first fermentation stage, the second fermentation stage and the third fermentation stage.

[0043] In the present invention, the temperature of the fermentation culture is preferably 35-38°C, more preferably 35-36°C; the rotation speed is preferably 150-200rpm, more preferably 150-160rpm; the fermentation culture time is preferably 28-30h, more preferably 29h. The pH value during the fermentation culture is preferably 6.9-7.1, more preferably 7.0. Liquid ammonia is introduced during the fermentation culture to maintain the pH value at 6.9-7.1.

[0044] The pressure of the fermentation tank used in the fermentation culture is preferably 0.03-0.05 MPa, more preferably 0.04 MPa, and the ventilation rate of the fermentation culture is 0.05-0.2 m3 / min per cubic meter of fermentation liquid. 3 Air, preferably 0.10-0.15 m3 of air per minute per cubic meter of fermentation liquid. 3Air. The fermentation medium of the present invention uses water as a solvent, and preferably includes the following components in concentrations: 3-5 g / L yeast powder, 2-3 g / L corn steep liquor, 1-1.2 g / L betaine, 10-15 g / L glucose, 0.2-0.5 g / L magnesium sulfate, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L ammonium citrate, 3-6 mg / L cobalt chloride, and 20-25 mg / L nicotinamide, and more preferably 5 g / L yeast powder, 2.5 g / L corn steep liquor, 1.1 g / L betaine, 13 g / L glucose, 0.3 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 3 g / L ammonium citrate, 4 mg / L cobalt chloride, and 22 mg / L nicotinamide.

[0045] In the present invention, the inoculation method of the Escherichia coli preferably includes seed liquid; the inoculation amount of the seed liquid is preferably 13% to 15% of the volume of the fermentation medium, and more preferably 14%. The present invention has no special limitation on the inoculation method, and conventional methods can be used. The preparation method of the seed liquid preferably includes: inoculating Escherichia coli into a seed culture medium for seed culture to obtain the Escherichia coli seed liquid; the OD of the seed liquid 600 The value is 10-12, more preferably 10-11. The OD of the Escherichia coli seed solution of the present invention is 600 When the value is 10-12, Escherichia coli is in the logarithmic growth phase and has the advantage of rapid growth and reproduction. The temperature of the seed culture is preferably 35-37°C, more preferably 36°C, the pH value during the seed culture is 6.9-7.0, and the pressure of the seed culture is preferably 0.03-0.05MPa, more preferably 0.04MPa. The seed culture medium of the present invention uses water as a solvent, and preferably includes the following concentrations of components: yeast powder 8-10g / L, glucose 25-30g / L, magnesium sulfate 0.5-0.8g / L, dipotassium hydrogen phosphate 2-4g / L, potassium dihydrogen phosphate 2-4g / L and ammonium sulfate 6-8g / L, more preferably including the following concentrations of components: yeast powder 9g / L, glucose 27g / L, magnesium sulfate 0.7g / L, dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 3g / L, ammonium sulfate 8g / L.

[0046] Generally, the stirring direction during fermentation production is clockwise, which will press down the fermentation liquid to avoid the situation where the foam increases during the fermentation process and affects the fermentation. However, during the fermentation of L-valine, the consumption rate of ammonium ions is slow, and the rate of liquid ammonia introduced is greater than the consumption rate. If the fermentation liquid is pressed down, the free ammonium ions in the fermentation liquid cannot be discharged from the fermentation liquid in time, the ammonia nitrogen content is getting higher and higher, the L-valine conversion rate is low, and the L-valine yield is low.

[0047] Therefore, in order to solve the above technical problems, the present invention produces L-valine by fermenting by controlling the ammonia nitrogen content in the fermentation liquid. At the beginning of the fermentation culture, the stirring direction is set to rotate counterclockwise, and the fermentation liquid rises so that excess free ammonium ions are quickly discharged from the fermentation tank along with the air, thereby preventing the ammonia nitrogen content from increasing rapidly. As the fermentation culture time increases, the ammonia nitrogen content becomes higher and higher, and the stirring direction is set to rotate clockwise, and by adding glucose solutions of different pH values, the ammonia nitrogen content in the fermentation liquid is controlled to be within 0.2% (w / v), thereby effectively reducing the synthesis of other miscellaneous acids such as organic acids and other amino acids, and improving the conversion rate of L-valine.

[0048] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] The same strain of Escherichia coli is used in the following examples and comparative examples of the present invention. The present invention has no particular limitation on the strain type of the Escherichia coli, and any strain type of Escherichia coli conventionally used for fermentation to prepare L-valine in the art can be used.

[0050] The pH adjuster of glucose solution is potassium hydroxide.

[0051] Example 1

[0052] A fermentation method for reducing the content of heteroacids in L-valine fermentation broth, comprising the following steps:

[0053] (1) Escherichia coli was inoculated into the seed culture medium for seed culture. The temperature of the seed culture was 36°C, the pH value during the seed culture process was 7.0, and the tank pressure of the seed tank used for seed culture was 0.05 MPa. When OD 600 When the value is 10, the culture is completed and the Escherichia coli seed solution is obtained; wherein, the seed culture medium uses water as solvent, and the component concentrations are as follows: yeast powder 8g / L, glucose 25g / L, magnesium sulfate 0.5g / L, dipotassium hydrogen phosphate 2g / L, potassium dihydrogen phosphate 2g / L and ammonium sulfate 6g / L.

[0054] (2) The fermentation medium was inoculated with seed liquid at a volume ratio of 14% for fermentation. The fermentation temperature was controlled at 35°C, the pH value was controlled at 7.1 during the entire fermentation process, the stirring speed was 150 rpm, the pressure was 0.03 MPa, and the ventilation volume was 0.15 m3 / min per cubic meter of fermentation liquid. 3 When the glucose concentration in the fermentation broth was ≤0.2% (w / v), glucose solution was added to control the glucose concentration in the fermentation broth to be 0.15% (w / v).

[0055] The fermentation medium uses water as solvent, and the component concentrations are as follows: yeast powder 3g / L, corn steep liquor 2g / L, betaine 1g / L, glucose 10g / L, magnesium sulfate 0.2g / L, potassium dihydrogen phosphate 1g / L, ammonium citrate 2g / L, cobalt chloride 3mg / L and nicotinamide 20mg / L.

[0056] During the fermentation culture, the ammonia nitrogen content in the fermentation liquid was detected every 2 hours. The fermentation culture consisted of a first fermentation stage, a second fermentation stage and a third fermentation stage, and the first fermentation stage, the second fermentation stage and the third fermentation stage were carried out in sequence.

[0057] The first fermentation stage: when the ammonia nitrogen content in the fermentation liquid is ≤0.12% (w / v), the stirring direction is set to counterclockwise rotation, and 60% (w / v) glucose solution with a pH value of 6.0~7.0 is added.

[0058] Second fermentation stage: When the ammonia nitrogen content in the fermentation liquid is greater than 0.12% and less than 0.15% (w / v), the stirring direction is restored from the counterclockwise rotation in the first fermentation stage to clockwise rotation, and 60% (w / v) glucose solution with a pH value of 9.0 is added.

[0059] The third fermentation stage: when the ammonia nitrogen content in the fermentation liquid is ≥0.15% (w / v), the stirring direction is clockwise and 60% (w / v) glucose solution with a pH value of 10.0 is added.

[0060] As the third fermentation stage progresses, the ammonia nitrogen content in the fermentation liquid decreases to the range of the second fermentation stage. After the ammonia nitrogen content decreases, the stirring direction and the addition of glucose solution are adjusted according to the requirements of the second fermentation stage until the fermentation culture time is 28 hours and the fermentation is completed.

[0061] The results of fermentation index determination are shown in Table 1.

[0062] Example 2

[0063] A fermentation method for reducing the content of heteroacids in L-valine fermentation broth, comprising the following steps:

[0064] (1) Escherichia coli was inoculated into the seed culture medium for seed culture. The temperature of the seed culture was controlled at 37°C, the pH value during the seed culture process was 6.9, and the tank pressure of the seed tank used for seed culture was 0.03 MPa. When the OD 600 When the value is 11, the culture is completed and the Escherichia coli seed solution is obtained; the seed culture medium uses water as the solvent, and the component concentrations are as follows: yeast powder 9g / L, glucose 27g / L, magnesium sulfate 0.7g / L, dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 3g / L and ammonium sulfate 8g / L.

[0065] (2) The fermentation medium was inoculated with seed liquid at a volume ratio of 15% for fermentation. The fermentation temperature was controlled at 36°C, the pH value was controlled at 7.0 during the entire fermentation process, the stirring speed was 180 rpm, the pressure was 0.05 MPa, and the ventilation volume was 0.10 m3 / min per cubic meter of fermentation liquid. 3 When the glucose concentration in the fermentation broth was ≤0.2% (w / v), glucose solution was added to control the glucose concentration in the fermentation broth to be 0.10% (w / v).

[0066] The fermentation medium uses water as solvent, and the component concentrations are as follows: yeast powder 5g / L, corn steep liquor 2.5g / L, betaine 1.1g / L, glucose 13g / L, magnesium sulfate 0.3g / L, potassium dihydrogen phosphate 1.3g / L, ammonium citrate 3g / L, cobalt chloride 4mg / L and nicotinamide 22mg / L.

[0067] During the fermentation culture, the ammonia nitrogen content in the fermentation liquid was detected every 2 hours. The fermentation culture consisted of a first fermentation stage, a second fermentation stage and a third fermentation stage, and the first fermentation stage, the second fermentation stage and the third fermentation stage were carried out in sequence.

[0068] The first fermentation stage: when the ammonia nitrogen content in the fermentation liquid is ≤0.12% (w / v), the stirring direction is set to counterclockwise rotation, and 62% (w / v) glucose solution with a pH value of 6.0~7.0 is added.

[0069] In the second fermentation stage, when the ammonia nitrogen content in the fermentation liquid is greater than 0.12% and less than 0.15%, the stirring direction is restored from the counterclockwise rotation in the first fermentation stage to clockwise rotation, and 62% (w / v) glucose solution with a pH value of 9.2 is added.

[0070] In the third fermentation stage, when the ammonia nitrogen content in the fermentation liquid is ≥0.15% (w / v), the stirring direction is clockwise, and 62% (w / v) glucose solution with a pH value of 10.2 is added.

[0071] As the third fermentation stage progressed, the ammonia nitrogen content in the fermentation liquid decreased to the range of the second fermentation stage. After the ammonia nitrogen content decreased, the stirring direction and the addition of glucose solution were adjusted according to the requirements of the second fermentation stage until the fermentation culture time was 29 hours and the fermentation was terminated. The results of the fermentation index determination are shown in Table 1.

[0072] Comparative Example 1

[0073] The preparation method of L-valine is the same as (1) to (2) of Example 1, with the only difference being that the stirring direction during the entire fermentation process in step (2) is clockwise.

[0074] Comparative Example 2

[0075] The preparation method of L-valine is the same as (1) to (2) of Example 1, with the only difference being that the stirring direction during the entire fermentation process in step (2) is counterclockwise.

[0076] Comparative Example 3

[0077] The preparation method of L-valine is the same as (1) to (2) of Example 1, with the only difference being that in step (2), 60% (w / v) glucose solution is fed during the entire fermentation process, and the pH value of the glucose solution is 6.0 to 7.0.

[0078] Comparative Example 4

[0079] The preparation method of L-valine is the same as (1) to (2) of Example 1, with the only difference being that in step (2), 60% (w / v) glucose solution is added during the entire fermentation process, and the pH value of the glucose solution is 9.0.

[0080] Comparative Example 5

[0081] The preparation method of L-valine is the same as (1) to (2) of Example 1, the only difference being that in step (2), 60% (w / v) glucose solution is added during the entire fermentation process, and the pH value of the glucose solution is 10.0.

[0082] Comparative Example 6

[0083] The preparation method of L-valine is the same as (1) to (2) of Example 1, with the only difference being that step (2) is carried out according to the traditional fermentation method, the stirring direction is clockwise during the entire fermentation process, 60% (w / v) glucose solution is fed, the pH value of the glucose solution is 6.0-7.0, and the fermentation culture time is 30 h.

[0084] The ammonia nitrogen content of the fermentation broth during the fermentation culture of Examples 1 to 2 and Comparative Examples 1 to 6 was detected, and the ammonia nitrogen content in the fermentation broth was detected by sampling every 2 hours, and the detection method was the Kjeldahl method.

[0085] Due to the large number of pictures, the present invention only shows the content of ammonia nitrogen in the fermentation broth in the fermentation culture in Example 1 and Comparative Example 6. The content of ammonia nitrogen in the fermentation broth in the fermentation culture in Example 1 is as follows: Figure 1 As shown, the ammonia nitrogen content in the fermentation broth of Comparative Example 6 is as follows Figure 2 As shown. Example 1 In the early stage of fermentation, as the culture time increases, the ammonia nitrogen content in the fermentation broth increases slowly. When the cycle is about 8 hours, the ammonia nitrogen content increases to 0.12% (w / v). At this time, glucose solutions with different pH values ​​are added, and the ammonia nitrogen content continues to increase slowly to a maximum of 0.17% (w / v), and then slowly decreases to 0.12%~0.15% (w / v). Figure 2It can be seen that according to the traditional fermentation method of Comparative Example 6, the ammonia nitrogen content increased to 0.12% (w / v) when the fermentation time was about 4 hours, and as the culture time increased, the ammonia nitrogen content in the fermentation liquid continued to increase until the ammonia nitrogen content was 0.45% (w / v) at the end of the fermentation.

[0086] The above results indicate that the method of the present invention can effectively control the ammonia nitrogen content in the fermentation broth.

[0087] The contents of L-valine and heteroacids in the fermentation broth at the end of the fermentation culture of Examples 1 to 2 and Comparative Examples 1 to 6 were detected. The contents of L-valine and heteroacids were determined using an amino acid analyzer, wherein the L-valine content was determined by an external standard method, and the heteroacid content was determined by a peak area normalization method.

[0088] The conversion rate is calculated as follows: Conversion rate = L-valine production / glucose production 100%, where L-valine production refers to L-valine content The discharge volume refers to the volume of all materials in the fermentation tank after the fermentation is completed, and the glucose amount is the total amount of glucose used in the fermentation process.

[0089] The results of fermentation index determination are shown in Table 1.

[0090] Table 1 The results of the determination of the fermentation indexes in Examples 1 to 2 and Comparative Examples 1 to 6

[0091]

[0092] As can be seen from Table 1, in Example 1 and Example 2, the stirring direction and the pH value of the glucose addition were adjusted according to the ammonia nitrogen content in the fermentation broth at different fermentation periods, so that the ammonia nitrogen content in the fermentation broth was controlled within a reasonable range (0.04%~0.2%), and the content of heteroacids in the fermentation broth was low at the end of fermentation, and the conversion rate was high.

[0093] In comparative example 1, the stirring direction was controlled to rotate clockwise throughout the whole process. The ammonia nitrogen content increased rapidly in the early stage of fermentation, but after the addition of glucose solutions with different pH values ​​in the later stage, the ammonia nitrogen content was within a reasonable range, the L-valine content was low, and the miscellaneous acid content was high.

[0094] In Comparative Example 2, the stirring direction was controlled to rotate counterclockwise throughout the whole process. In the middle and late stages of fermentation, as the volume of the fermentation liquid increased, the microorganisms grew and multiplied, the foam in the fermenter was large, and the fermentation liquid overflowed seriously, causing contamination, and the tank was stopped halfway.

[0095] In comparative example 3, the pH value of the 60% glucose solution added throughout the process was 6.0-7.0. Although the ammonia nitrogen content increased slowly in the early stage due to the counterclockwise rotation, with the rapid growth and reproduction of microorganisms in the later stage, the frequency of liquid ammonia introduction gradually increased, and the ammonia nitrogen content in the fermentation liquid increased rapidly, reaching a maximum of 0.40%.

[0096] The pH values ​​of the 60% (w / v) glucose solutions added throughout the entire process in Comparative Examples 4 and 5 were 9.0 and 10.0. The frequency of liquid ammonia introduction decreased during the fermentation process. As the microorganisms grew and multiplied, the ammonium ions in the fermentation broth were rapidly utilized, causing the ammonia nitrogen content in the fermentation broth to gradually decrease. In the late stage of fermentation, ammonia nitrogen was seriously insufficient, and the acid production of the bacteria was inhibited.

[0097] Comparative Example 6 follows the traditional fermentation method and does not control the ammonia nitrogen content in the fermentation liquid. At the end of the fermentation, the ammonia nitrogen content reaches 0.45%, the impurity acid content is high, and the conversion rate is low.

[0098] It can be seen that the technical solution of the present invention has the following advantages compared with the comparative example: by controlling different stirring directions and adding glucose solutions with different pH values ​​according to the ammonia nitrogen content in the fermentation broth, the ammonia nitrogen content in the fermentation broth is controlled within the optimal range suitable for high production of L-valine by Escherichia coli, the content of miscellaneous acids in the fermentation broth is reduced, and the conversion rate of L-valine is improved.

[0099] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fermentation method for reducing the content of heteroacids in L-valine fermentation broth, characterized in that: The steps include: Escherichia coli was inoculated into the fermentation medium for fermentation culture. When the glucose concentration in the fermentation liquid was ≤0.2% w / v, glucose solution was added. After the fermentation was completed, a fermentation liquid containing L-valine was obtained. The ventilation rate of the fermentation culture was 0.05-0.2 m3 / min per cubic meter of fermentation liquid. 3 air; the mass concentration of the glucose solution is 60% to 65%; after the glucose solution is added, the glucose concentration in the fermentation liquid is controlled to be 0.1% to 0.2% w / v; During the fermentation culture, when the ammonia nitrogen content in the fermentation liquid is ≤0.12% w / v, the stirring direction is counterclockwise rotation and a glucose solution with a pH value of 6.0-7.0 is added; the counterclockwise rotation will raise the fermentation liquid; When the ammonia nitrogen content in the fermentation liquid is greater than 0.12% w / v and less than 0.15% w / v, the stirring direction is clockwise rotation and a glucose solution with a pH value of 9.0 to 9.2 is added; the clockwise rotation presses down the fermentation liquid; When the ammonia nitrogen content in the fermentation broth is ≥0.15% w / v, the stirring direction is clockwise and a glucose solution with a pH value of 10.0-10.2 is added; when the ammonia nitrogen content in the fermentation broth drops to greater than 0.12% and less than 0.15%, the stirring direction is clockwise and a glucose solution with a pH value of 9.0-9.2 is added; The fermentation medium uses water as a solvent and includes components in the following concentrations: 3-5 g / L yeast powder, 2-3 g / L corn steep liquor, 1-1.2 g / L betaine, 10-15 g / L glucose, 0.2-0.5 g / L magnesium sulfate, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L ammonium citrate, 3-6 mg / L cobalt chloride and 20-25 mg / L nicotinamide.

2. The fermentation method for reducing the content of heteroacids in L-valine fermentation broth according to claim 1, characterized in that: The fermentation temperature is 35-38°C, the rotation speed is 150-200 rpm, the fermentation time is 28-30 hours, the fermentation pressure is 0.03-0.05 MPa, and the pH value during the fermentation process is 6.9-7.

1.

3. The fermentation method for reducing the content of heteroacids in L-valine fermentation broth according to claim 1, characterized in that: The inoculation method of the Escherichia coli includes seed liquid; the inoculation amount of the seed liquid is 13% to 15% of the volume of the fermentation medium.

4. The fermentation method for reducing the content of heteroacids in L-valine fermentation broth according to claim 3, characterized in that: The OD of the seed solution 600 The value is 10~12.

5. The fermentation method for reducing the content of heteroacids in L-valine fermentation broth according to claim 3 or 4, characterized in that: The preparation method of the seed liquid comprises: inoculating Escherichia coli into a seed culture medium for seed culture to obtain the Escherichia coli seed liquid; the seed culture medium uses water as a solvent and further comprises components of the following concentrations: 8-10 g / L yeast powder, 25-30 g / L glucose, 0.5-0.8 g / L magnesium sulfate, 2-4 g / L dipotassium hydrogen phosphate, 2-4 g / L potassium dihydrogen phosphate and 6-8 g / L ammonium sulfate.

6. The fermentation method for reducing the content of heteroacids in L-valine fermentation broth according to claim 5, characterized in that: The temperature of the seed culture is 35-37° C., the pH value during the seed culture process is 6.9-7.0, and the pressure of the seed culture is 0.03-0.05 MPa.

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