A method for shortening the fermentation cycle of L-isoleucine

Abstract

This invention provides a method for shortening the fermentation cycle of L-isoleucine, belonging to the field of amino acid fermentation technology. The method involves inoculating L-isoleucine-producing bacteria into a fermentation medium and culturing until the sugar solution in the fermentation system is depleted and dissolved oxygen and pH levels show a rebound. This invention, by adding nutrients in a single step during L-isoleucine fermentation, eliminates the need for continuous addition of sugar solution and other nutrients during fermentation. This extends the logarithmic phase of the L-isoleucine-producing bacteria, resulting in massive bacterial proliferation and effectively shortening the fermentation cycle by 30%–40%. Simultaneously, by adjusting the nutrient content, the yield and conversion rate of L-isoleucine are maintained.

Description

Technical Field

[0001] This invention belongs to the field of amino acid fermentation technology, specifically relating to a method for shortening the fermentation cycle of L-isoleucine. Background Technology

[0002] L-Isoleucine, also known as isoleucine or 2-amino-3-methylpentanoic acid, has the chemical formula C6H12H2O. 13 NO2 is one of the essential amino acids for the human body. L-Isoleucine has important applications in the pharmaceutical, health, and food industries.

[0003] Currently, there are three main methods for producing L-isoleucine: extraction, chemical synthesis, and fermentation. Extraction and chemical synthesis methods are unsuitable for industrial production due to difficulties in separating L-isoleucine from its isomers, limited raw material sources, high production costs, and environmental pollution. Fermentation, on the other hand, offers mild conditions, is environmentally friendly, and produces stable product quality, making it the primary method for L-isoleucine production. However, existing fermentation methods for L-isoleucine production generally have excessively long fermentation cycles due to the fermentation process itself. These extended fermentation cycles increase production costs in terms of electricity, gas, and labor, and also increase the risk of microbial contamination during fermentation. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for shortening the fermentation cycle of L-isoleucine, which can both ensure the yield and conversion rate of L-isoleucine and shorten the fermentation cycle.

[0005] This invention provides a method for shortening the fermentation cycle of L-isoleucine, comprising the following steps:

[0006] Corynebacterium glutamate ( Corynebacterium glutamicum The bacterial culture is inoculated into the fermentation medium and fermented until the dissolved oxygen and pH value in the fermentation system show a rebound.

[0007] The fermentation medium comprises the following components in the following proportions: glucose 120-210 g / L, ammonium sulfate 2-3 g / L, magnesium sulfate 0.25-1 g / L, beet molasses 15-20 g / L, ferrous sulfate 1.8-2.5 mg / L, manganese sulfate 2-3.5 mg / L, potassium dihydrogen phosphate 0.55-1.0 g / L, copper sulfate 0.3-0.6 mg / L, zinc sulfate 0.4-0.7 mg / L, biotin 6-10 mg / L, and a vitamin mixture 20-80 mg / L.

[0008] Preferably, the fermentation medium comprises the following components in the following amounts: glucose 125~167 g / L, ammonium sulfate 2.2~2.8 g / L, magnesium sulfate 0.39~0.8 g / L, beet molasses 17~19 g / L, ferrous sulfate 1.95~2.15 mg / L, manganese sulfate 2.5~3.5 mg / L, potassium dihydrogen phosphate 0.7~1.0 g / L, copper sulfate 0.45~0.55 mg / L, zinc sulfate 0.55~0.65 mg / L, biotin 7.5~8.5 mg / L, and a vitamin mixture 60~70 mg / L.

[0009] Preferably, the initial dissolved oxygen rate of the fermentation medium is 80%~85%; the fermentation temperature is 31~33℃; and the fermentation pressure is 0.03~0.15 MPa.

[0010] Preferably, during fermentation, the dissolved oxygen rate of the fermentation system is controlled at 20% to 30% of the initial dissolved oxygen; and the pH value of the fermentation system is controlled at 7.2 to 7.4.

[0011] Preferably, the method for controlling the dissolved oxygen rate of the fermentation system is to adjust the stirring speed and air volume;

[0012] The stirring speed is 300~600 rpm, and the air volume is 0.6~1.3 m³ / h.

[0013] Preferably, the inoculation amount of the Corynebacterium glutamicum bacterial suspension is 13% to 15%.

[0014] Preferably, the primary seed culture medium for culturing the Corynebacterium glutamicum bacterial suspension comprises the following components: glucose 60-70 g / L, magnesium sulfate 1-2 g / L, beet molasses 25-30 g / L, corn steep liquor 30-50 g / L, ferrous sulfate 0.04-0.06 g / L, manganese sulfate 0.02-0.05 g / L, potassium dihydrogen phosphate 3.5-5.5 g / L, copper sulfate 1-2 mg / L, zinc sulfate 0.6-0.8 mg / L, biotin 0.03-0.04 g / L, and a vitamin mixture 0.05-0.06 g / L; the culture temperature is 30-31°C.

[0015] Preferably, the initial dissolved oxygen rate of the primary seed culture medium is 100%; the culture pressure is 0.05 MPa; and the culture temperature is 30~31℃.

[0016] Preferably, during the cultivation of the L-isoleucine-producing bacterial culture, the dissolved oxygen rate of the culture system is controlled at 30% to 40%, and the pH value of the culture system is controlled at 7.2 to 7.4.

[0017] Preferably, the Corynebacterium glutamicum includes Corynebacterium glutamicum strain DXMC601; the preservation number of Corynebacterium glutamicum strain DXMC601 is CCTCC NO: M 2025347.

[0018] This invention provides a method for shortening the fermentation cycle of L-isoleucine, comprising the following steps: feeding Corynebacterium glutamicum (… Corynebacterium glutamicum The bacterial culture is inoculated into the fermentation medium for fermentation. The fermentation is stopped when the sugar in the fermentation system is depleted and the dissolved oxygen and pH value rise again. The fermentation medium includes the following components: glucose 120~210g / L, ammonium sulfate 2~3g / L, magnesium sulfate 0.25~1g / L, beet molasses 15~20g / L, ferrous sulfate 1.8~2.5mg / L, manganese sulfate 2~3.5mg / L, potassium dihydrogen phosphate 0.55~1.0g / L, copper sulfate 0.3~0.6mg / L, zinc sulfate 0.4~0.7mg / L, biotin 6~10mg / L, and vitamin mixture 20~80mg / L. This invention addresses the problem of prolonged fermentation cycles caused by the addition of sugar solutions and other nutrients in traditional L-isoleucine fermentation. It creatively adds the base sugar and other nutrients to the fermentation medium in a single step, significantly extending the logarithmic growth phase of the L-isoleucine-producing bacteria, leading to mass cell proliferation and shortening the fermentation cycle by 30%–40%. Furthermore, by optimizing the amounts of components added to the fermentation medium, this invention achieves both a shortened fermentation cycle and high L-isoleucine yield while maintaining a high conversion rate. Therefore, the technical solution provided by this invention significantly shortens the fermentation cycle, reduces electricity consumption and costs, minimizes the risk of contamination during fermentation, and simultaneously ensures high L-isoleucine yield and conversion rate.

[0019] Instructions for the Preservation of Biological Materials

[0020] Corynebacterium glutamicum ( Corynebacterium glutamicum Strain DXMC601 is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The deposit date is March 3, 2025, and the accession number is CCTCC NO: M 2025347. Detailed Implementation

[0021] This invention provides a method for shortening the fermentation cycle of L-isoleucine, comprising the following steps:

[0022] Corynebacterium glutamate ( Corynebacterium glutamicum The bacterial culture is inoculated into the fermentation medium for fermentation culture. The fermentation is stopped when the dissolved oxygen and pH value in the fermentation system show a rebound.

[0023] In this invention, addressing the problem of prolonged fermentation cycles caused by traditional fed-batch fermentation, the method involves adding base sugars and other nutrients all at once to form a fermentation medium, omitting the fed-batch step. This allows L-isoleucine-producing bacteria to proliferate rapidly in a short period. Simultaneously, the amount of added medium components is adjusted to meet the metabolic needs of the L-isoleucine-producing bacteria, thereby enabling large-scale fermentation and production of L-isoleucine. This shortens the fermentation cycle while ensuring the yield and conversion rate of L-isoleucine.

[0024] In this invention, the method for preparing the Corynebacterium glutamicum bacterial suspension preferably involves inoculating Corynebacterium glutamicum into a primary culture medium and culturing it. In an embodiment of this invention, Corynebacterium glutamicum with accession number CCTCC NO: M 2025347 ( Corynebacterium glutamicum The strain DXMC601 is used as an L-isoleucine producing bacterium for L-isoleucine fermentation to illustrate the technical effects of the present invention.

[0025] In this invention, the primary seed culture medium preferably comprises the following components: glucose 60-70 g / L, ammonium sulfate 2-3 g / L, magnesium sulfate 1-2 g / L, beet molasses 25-30 g / L, corn steep liquor 30-50 g / L, ferrous sulfate 0.04-0.06 g / L, manganese sulfate 0.02-0.05 g / L, potassium dihydrogen phosphate 3.5-5.5 g / L, copper sulfate 1-2 mg / L, zinc sulfate 0.6-0.8 mg / L, biotin 0.03-0.04 g / L, and a vitamin mixture 0.05-0.06 g / L. The vitamin mixture preferably comprises VB1, VB3, and VB5. The mass ratio of VB1, VB3, and VB5 is preferably 1.5-2.5:0.8-1.2:1.5-2.5, but can also be 2:1:2. The preferred method for preparing the primary seed culture medium is to separately sterilize a mixture of glucose, magnesium sulfate, and beet molasses, while dissolving and bringing the volume to a final volume in a fermenter for sterilization. The separately sterilized mixture of glucose, magnesium sulfate, and beet molasses is then combined with the remaining components. The preferred sterilization conditions for separate sterilization are 118–121°C for 20–30 min. The preferred sterilization conditions for final sterilization are 121–122°C for 20–30 min. The primary seed culture medium also preferably includes an antifoaming agent. The preferred concentration of the antifoaming agent is 0.08–0.1 ml / L. This invention does not impose any particular limitation on the type of antifoaming agent; any antifoaming agent well-known in the art can be used. The culture is preferably carried out in a fermenter. A 30L fermenter is preferably used to hold 13–14L of the primary seed culture medium.

[0026] In this invention, the initial dissolved oxygen rate of the primary seed culture medium is preferably 100%. The preferred method for adjusting the initial dissolved oxygen rate of the primary seed culture medium is to adjust it by adjusting the stirring speed and ventilation rate. Specifically, the preferred stirring speed is 600 rpm, the tank pressure is 0.05 MPa, the ventilation rate is 12 L / min, and the condition is maintained stable for 5 minutes. The preferred culture conditions for the L-isoleucine-producing bacterial solution are: culture pressure 0.05 MPa, dissolved oxygen rate controlled at 30%–40%, pH value controlled at 7.2–7.4, and culture temperature 30–31°C. When the dissolved oxygen rate of the culture system decreases below 30% during the culture process, the dissolved oxygen is adjusted back to the above-mentioned suitable range by increasing the stirring speed and ventilation rate.

[0027] In this invention, the culture is preferably carried out until the OD value of the L-isoleucine-producing bacterial solution reaches 0.6-0.7, indicating that the bacterial solution has entered the logarithmic phase. The bacterial cells in the logarithmic phase have high activity and are suitable for fermentation culture. The OD value is preferably measured using a visible spectrophotometer at a wavelength of 562 nm and a 50-fold dilution.

[0028] In this invention, before fermentation, the L-isoleucine-producing bacterial culture is inoculated into the fermentation medium. The inoculation amount of the L-isoleucine-producing bacterial culture is preferably 13% to 15%, and can be 14%.

[0029] In this invention, the fermentation medium preferably comprises the following components in the following amounts: glucose 125-167 g / L, magnesium sulfate 0.39-0.8 g / L, beet molasses 17-19 g / L, ferrous sulfate 1.95-2.15 mg / L, manganese sulfate 2.5-3.5 mg / L, potassium dihydrogen phosphate 0.7-1.0 g / L, copper sulfate 0.45-0.55 mg / L, zinc sulfate 0.55-0.65 mg / L, biotin 7.5-8.5 mg / L, and a vitamin mixture 60-70 mg / L. The vitamin mixture preferably comprises VB1, VB3, and VB5. The mass ratio of VB1, VB3, and VB5 is preferably 2.5-3.5:0.8-1.2:2.5-3.5, and can be 3:1:3. The preferred method for preparing the fermentation medium is to separately sterilize glucose, magnesium sulfate, and beet molasses after dissolving them in a mixture, while the remaining components of the fermentation medium are mixed and dissolved in a fermenter for sterilization. The preferred sterilization conditions for separate sterilization are 118-121°C for 20-30 minutes. The preferred sterilization conditions for the actual sterilization are 121-122°C for 20-30 minutes. Before inoculation, the separately sterilized glucose, magnesium sulfate, and beet molasses are mixed with the other components of the fermentation medium. The fermentation is preferably carried out in a fermenter. The preferred volume of the fermentation medium in a 50L fermenter is 24-25L.

[0030] In this invention, before inoculation, the initial dissolved oxygen rate of the fermentation medium is 80%~85%, which can be 81%~84% or 82%~83%. The preferred method for calibrating the dissolved oxygen of the fermentation medium is to maintain a stable dissolved oxygen rate of 100% for 5 minutes at a temperature of 32℃, pH 7.4, stirring speed of 600 rpm, tank pressure of 0.05 MPa, and airflow of 1 m³ / h. During the fermentation process, the preferred fermentation temperature is 31~33℃, which can be 32℃; the preferred pressure of the fermentation system is 0.03~0.15 MPa, which can be 0.05~0.12 MPa or 0.08~0.1 MPa; the preferred stirring speed is 300~600 rpm, which can be 400~500 rpm or 450 rpm; and the preferred airflow is 0.6~1.3 m³ / L, which can be 0.6~1.2 m³ / L or 0.7~0.8 m³ / L. As fermentation progresses, dissolved oxygen in the fermentation system gradually decreases. When the dissolved oxygen level drops below 20%, the stirring speed and airflow are gradually increased to maintain the dissolved oxygen level at 20%–30%, while keeping the tank pressure constant. If dissolved oxygen becomes too high in the later stages of fermentation, the stirring speed is reduced while maintaining a constant airflow. The OD value, residual sugar, ammonia nitrogen, and acid content of the bacterial solution are monitored periodically during fermentation. Fermentation is terminated after a 10-minute pH and dissolved oxygen reversal.

[0031] In this invention, to further demonstrate the advantages of the method, a fed-batch fermentation method was also used for L-isoleucine fermentation. Experiments showed that, to achieve the same or similar L-isoleucine yield and conversion rate as this invention, the fermentation cycle required 36 hours, while this invention only required 21-30 hours. Furthermore, artificially controlling the fermentation time to 24 hours using the fed-batch method significantly reduced the yield and conversion rate of L-isoleucine. In addition, Comparative Example 6, while shortening the fermentation cycle by improperly adjusting the composition of the fermentation medium, reduced the conversion rate of L-isoleucine; therefore, this fermentation method is not conducive to reducing production costs.

[0032] The following detailed description of a method for shortening the fermentation cycle of L-isoleucine provided by the present invention, with reference to specific embodiments, should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] A method for shortening the fermentation cycle of L-isoleucine includes the following steps:

[0035] Prepare the primary seed culture medium: Accurately weigh the following reagents: 0.8g ferrous sulfate, 0.5g manganese sulfate, 60g potassium dihydrogen phosphate, 600g corn steep liquor, 18mg copper sulfate, 11mg zinc sulfate, 0.5g biotin, 0.7g vitamin mixture (VB1, VB3, and VB5 in a mass ratio of 2:1:2), and 2ml bubbly agent. Dissolve the reagents in water and bring the volume to 11L in a 30L fermentation tank. Sterilize at 121℃ for 20 minutes, resulting in a final volume of 12.5L. Next, weigh 950g glucose, 23g magnesium sulfate, and 400g beet molasses, dissolve them in water, and bring the volume to 1.5L in a feed tank. Wrap the feed tube in gauze and kraft paper and sterilize in an autoclave at 121℃ for 20 minutes. After sterilization, use a peristaltic pump to add the solution from the feed tank to the seed tank. Set the temperature to 30℃, pH to 7.4, stirring speed to 600 rpm, tank pressure to 0.05 MPa, and airflow to 12 L / min. After maintaining stability for 5 minutes, calibrate the dissolved oxygen to 100%. Under flame protection, add 50 ml of seed culture (Corynebacterium glutamicum (…)). Corynebacterium glutamicum(Strain DXMC601, preservation number CCTCC NO: M 2025347) was poured into the primary seed tank through the inoculation port. The stirring speed was set to 300 rpm, the air volume to 6 L / min, the tank pressure to 0.05 MPa, and the temperature to remain constant. During fermentation, as dissolved oxygen decreased, the stirring speed and air volume were increased to maintain dissolved oxygen at 30%~40%. After fermentation continued for 8 hours, OD value was measured every 2 hours. The culture period was 22 hours, and the measured OD value was 0.653. The tank was then kept under pressure. Prepare the fermentation medium: Accurately weigh the following reagents: 50 mg ferrous sulfate, 70 mg manganese sulfate, 20 g potassium dihydrogen phosphate, 60 g ammonium sulfate, 12 mg copper sulfate, 14 mg zinc sulfate, 0.15 g biotin, 1.7 g of a vitamin mixture (VB1, VB3, and VB5 in a mass ratio of 3:1:3), and 2 ml of bubbly precipitate. Dissolve in water and bring the volume to 16 L in a 50 L fermenter. Sterilize at 121 °C for 20 min, resulting in a final volume of 18 L. Weigh 3 kg glucose, 19 g magnesium sulfate, and 430 g beet molasses. Dissolve in water and bring the volume to 6 L in a feed tank. Wrap the feeder in gauze and kraft paper and sterilize in an autoclave at 121 °C for 20 min. After sterilization, add the feed solution from the feed tank to the fermenter using a peristaltic pump. The temperature was set to 32℃, pH 7.4, stirring speed 600 rpm, tank pressure 0.05 MPa, and airflow 1 m³ / h. After maintaining stability for 5 minutes, dissolved oxygen was calibrated to 100%. The primary seed tank culture was transferred to the main tank via a transfer pipe, with a transfer volume of 4 L. The stirring speed was set to 300 rpm, tank pressure 0.05 MPa, and airflow 0.6 m³ / h to begin fermentation. During fermentation, the initial sugar content was 12 g / dL. As fermentation progressed, when dissolved oxygen fell below 20%, the stirring speed was gradually increased to maintain dissolved oxygen at 20%-30%. After increasing the stirring speed to 350 rpm, the airflow was increased by 0.8 m³ / h. The stirring speed was then gradually increased to 400 rpm, and the ventilation ratio was increased to 1.0 m³ / h, and so on, until the highest conditions were met. During the later stages of fermentation, when dissolved oxygen became high, the stirring speed was gradually reduced while the airflow remained constant until fermentation ended. During fermentation, the pH was adjusted with ammonia. OD value, residual sugar, and ammonia nitrogen were measured every 2 hours. Acid content was measured starting at 12 hours. OD was measured at a wavelength of 562 nm with a 200-fold dilution. Residual sugar was measured using a biosensor, ammonia nitrogen was measured by titration, and acid content was measured by liquid chromatography. The cycle was 21 hours. The pH rose, and after a 10-minute rise, the mixture was removed from the fermentation tank. Example 2

[0036] The primary seed culture medium and the tank control method are the same as in Example 1. The only difference between the fermentation culture medium and Example 1 is that the glucose mass is changed to 4 kg, while the tank control method is the same.

[0037] Example 3

[0038] The primary seed culture medium and the tank control method are the same as in Example 1. The only difference between the fermentation culture medium and Example 1 is that the glucose mass is changed to 5 kg, while the tank control method is the same.

[0039] Example 4

[0040] The primary seed culture medium and fermentation tank control method were the same as in Example 2, the only difference being that the magnesium sulfate content in the fermentation medium was reduced by 10%, down to 17g. The culture medium was dissolved in water and brought to a final volume of 16L in a 50L fermenter. Sterilization was performed at 121°C for 20 minutes, resulting in a final volume of 18L. 4kg of glucose powder, 17g of magnesium sulfate, and 430g of beet molasses were weighed and dissolved in water. The mixture was brought to a final volume of 6L in a feeding vessel. The plug was wrapped in gauze and kraft paper and placed in an autoclave for sterilization at 121°C for 20 minutes. The fermentation tank control method remained the same.

[0041] Example 5

[0042] The primary seed culture medium and fermentation tank control method were the same as in Example 2, the only difference being that the magnesium sulfate content in the fermentation medium was reduced by 20%, down to 15g. The culture medium was dissolved in water and brought to a final volume of 16L in a 50L fermenter. Sterilization was performed at 121°C for 20 minutes, resulting in a final volume of 18L. 4kg of glucose powder, 15g of magnesium sulfate, and 430g of beet molasses were weighed and dissolved in water. The mixture was brought to a final volume of 6L in a feeding vessel. The plug was wrapped in gauze and kraft paper and placed in an autoclave for sterilization at 121°C for 20 minutes. The fermentation tank control method remained the same.

[0043] Example 6

[0044] The culture medium and control method for the seed tank were the same as in Example 2, the only difference being that the amount of magnesium sulfate in the fermentation medium was reduced by 50%, down to 10g. The culture medium was dissolved in water and brought to a final volume of 16L in a 50L fermenter. Sterilization was performed at 121°C for 20 minutes, resulting in a final volume of 18L. 4kg of glucose powder, 10g of magnesium sulfate, and 430g of beet molasses were weighed and dissolved in water. The final volume was brought to a final volume of 6L in a feeding tank. The plug was wrapped in gauze and kraft paper and placed in an autoclave for sterilization at 121°C for 20 minutes. The control method remained the same.

[0045] Comparative Example 1

[0046] The culture medium and control method for the first seed tank are the same as in Example 1. The sugar at the bottom of the fermenter is no longer added all at once, but rather the sugar solution and other small ingredients are continuously added during the fermentation process. The fermentation medium components are: 8g potassium dihydrogen phosphate, 45g ammonium sulfate, 25mg ferrous sulfate, 40mg manganese sulfate, 10mg zinc sulfate, 10mg copper sulfate, 50mg biotin, and 1.7g of a vitamin mixture (VB1, VB3, and VB5 in a mass ratio of 3:1:3). After dissolving the materials, the mixture is brought to a final volume of 16L in a 50L fermenter and sterilized at 121℃ for 20 minutes, resulting in a final volume of 24L. 1kg of glucose powder, 15g of magnesium sulfate, and 300g of beet molasses are weighed, dissolved in water, and brought to a final volume of 1.5L in the addition tank 1. The plug is then wrapped with gauze and kraft paper. Prepare one sugar solution: Weigh 5 kg of glucose, 5 g of magnesium sulfate, and 140 g of beet molasses, dissolve in water, and bring the volume to 18 L in flow tank 2. Wrap the plug with gauze and kraft paper. Prepare one flow feed medium: The culture medium includes 20 mg of ferrous sulfate, 20 mg of manganese sulfate, 12 g of potassium dihydrogen phosphate, 2 mg of copper sulfate, 8 mg of zinc sulfate, and 80 mg of biotin. Dissolve the medium in water and bring the volume to 3 L in flow tank 3. Wrap the plug with gauze and kraft paper. Place flow tanks 1, 2, and 3 in an autoclave and sterilize at 121°C for 20 min.

[0047] After sterilization, the material from feed tank 1 was added to the fermenter in one go using a peristaltic pump. The temperature was set to 32℃, pH 7.4, stirring speed to 600 rpm, tank pressure to 0.05 MPa, and airflow to 1.3 m³ / h. After maintaining stability for 5 minutes, dissolved oxygen was calibrated to 100%. The primary seed tank culture was transferred to the fermenter via a transfer pipe, with a transfer volume of 4 L. Stirring speed was set to 250 rpm, tank pressure to 0.05 MPa, and ventilation ratio to 0.3, and fermentation was initiated under controlled conditions. After the pH rebounded, sugar solution and additives were added in a ratio of sugar solution:additives = 7:1 (mass ratio). Residual sugar was controlled at 0.2-0.3% throughout the process, and dissolved oxygen control was the same as in Example 1. The fermentation cycle was 40 hours.

[0048] Comparative Example 2

[0049] Comparative Example 2 had the same culture medium and fermentation conditions as Comparative Example 1. The difference was that Comparative Example 2 was in a continuous feed mode after the pH value returned to normal, but the fermentation cycle was 24 hours.

[0050] Comparative Example 3

[0051] Comparative Example 3 used the same culture medium and fermentation conditions as Comparative Example 1, except that the ratio of sugar solution to small feed in Comparative Example 3 was changed to 5:1, and the cycle was 36 hours.

[0052] Comparative Example 4

[0053] Comparative Example 4 used the same culture medium and fermentation conditions as Comparative Example 1, except that the ratio of sugar solution to feedstock in Comparative Example 4 was changed to 5:1, and the fermentation cycle was 24 hours.

[0054] Comparative Example 5

[0055] Comparative Example 5 used the same culture medium and fermentation conditions as Comparative Example 1, the difference being that the glucose content in the fed-batch sugar solution of Comparative Example 5 was increased by 50% (m / m), specifically, 10 kg of sugar powder was brought to a final volume of 18 L. The ratio of sugar solution to feedstock was changed to 5:1, and the fermentation cycle was 36 hours before being transferred to the tank.

[0056] Comparative Example 6

[0057] The seed tank culture medium and tank control method are the same as in Example 2. The only difference between the fermentation culture medium and Example 1 is that 100g of corn steep liquor is added to the fermentation culture medium. The tank control method is the same.

[0058] Comparative Example 7

[0059] The culture medium and control method in the seed tank were the same as in Example 2, the only difference being that the magnesium sulfate in the large tank culture medium was reduced by 70%, down to 5.7g. The culture medium was dissolved in water and brought to a final volume of 16L in a 50L fermenter. It was then sterilized at 121℃ for 20 minutes, resulting in a final volume of 18L. 4kg of glucose powder, 10g of magnesium sulfate, and 430g of beet molasses were weighed, dissolved in water, and brought to a final volume of 6L in a feeding tank. The plug was wrapped in gauze and kraft paper and placed in an autoclave for sterilization at 121℃ for 20 minutes. The control method remained the same.

[0060] The methods for measuring each index in Examples 1-6 and Comparative Examples 1-7 are as follows:

[0061] The dissolved oxygen rate of the fermentation system was measured using a Mettler dissolved oxygen electrode, showing 100% at the initial stage of fermentation.

[0062] OD value: Measured by a visible spectrophotometer at a wavelength of 562 nm;

[0063] pH value: Measured using a METTER OLEDO benchtop pH meter;

[0064] Residual reducing sugar content: determined using an SBA-40C enzyme membrane analyzer;

[0065] Ammonia nitrogen content: titrated with hydrochloric acid standard solution using methyl red-methylene blue as indicator;

[0066] L-Isoleucine content: determined by liquid chromatography;

[0067] Sugar-acid conversion rate = L-isoleucine production / total sugar consumption × 100% Formula I.

[0068] L-Isoleucine yield = Acid content × Lower tank volume Formula II;

[0069] The total sugar consumption is the actual mass of glucose consumed.

[0070] The measurement results of Examples 1-6 and Comparative Examples 1-6 are shown in Table 1.

[0071] Table 1. Fermentation cycle, cell count, L-isoleucine yield, and conversion rate results for different fermentation methods.

[0072]

[0073] The results of Examples 1-3 show that the OD value increases rapidly and the cycle is shortened by 30%-40% when the material is added all at once. The acid production and conversion rate are better than those of the continuous feed mode, but the initial sugar content should not be too high.

[0074] As can be seen from Examples 4-6, using a one-time feed-in mode reduces the amount of magnesium sulfate in the fermentation medium to 20%, extends the cycle by 3-4 hours, but improves acid production and conversion rate.

[0075] The results of Comparative Examples 1, 3, and 5 show that using the continuous feed-in mode, with different condition modifications, results in lower acid production and conversion rates compared to the one-time addition mode, and the cycle is significantly longer. The results of Comparative Examples 2 and 4 show that using the continuous feed-in mode, after 24 hours of feed-in fermentation, the acid content in the discharged material was significantly lower than in Example 1, and the conversion rate was also significantly reduced. The results of Comparative Example 6 show that in the one-time material addition mode, the addition of an extra nitrogen source nutrient resulted in a rapid increase in OD value and a further shortened fermentation cycle, but the conversion rate was significantly reduced. The results of Comparative Example 7 show that reducing the concentration of magnesium sulfate to a certain level reduces L-isoleucine production and sugar-acid conversion rate.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for shortening the fermentation cycle of L-isoleucine, characterized in that, Includes the following steps: Corynebacterium glutamate ( Corynebacterium glutamicum The bacterial culture is inoculated into the fermentation medium and fermented until the dissolved oxygen and pH value rise again after the sugar solution in the fermentation system is exhausted. The fermentation medium comprises the following components in the following proportions: glucose 120-210 g / L, ammonium sulfate 2-3 g / L, magnesium sulfate 0.25-1 g / L, beet molasses 15-20 g / L, ferrous sulfate 1.8-2.5 mg / L, manganese sulfate 2-3.5 mg / L, potassium dihydrogen phosphate 0.55-1.0 g / L, copper sulfate 0.3-0.6 mg / L, zinc sulfate 0.4-0.7 mg / L, biotin 6-10 mg / L, and a vitamin mixture 20-80 mg / L; The Corynebacterium glutamicum is Corynebacterium glutamicum strain DXMC601; The Corynebacterium glutamicum strain DXMC601 has the accession number CCTCC NO: M 2025347.

2. The method according to claim 1, characterized in that, The fermentation medium comprises the following components in the following amounts: glucose 125-167 g / L, ammonium sulfate 2.2-2.8 g / L, magnesium sulfate 0.39-0.8 g / L, beet molasses 17-19 g / L, ferrous sulfate 1.95-2.15 mg / L, manganese sulfate 2.5-3.3 mg / L, potassium dihydrogen phosphate 0.7-0.9 g / L, copper sulfate 0.45-0.55 mg / L, zinc sulfate 0.55-0.65 mg / L, biotin 7.5-8.5 mg / L, and a vitamin mixture 60-70 mg / L.

3. The method according to claim 1, characterized in that, The initial dissolved oxygen rate of the fermentation medium is 80%~85%; the fermentation temperature is 31~33℃; and the fermentation pressure is 0.03~0.15 MPa.

4. The method according to claim 1, characterized in that, The dissolved oxygen rate of the fermentation system is controlled at 20% to 30% of the initial dissolved oxygen; the pH value of the fermentation system is controlled at 7.2 to 7.

4.

5. The method according to claim 4, characterized in that, The method for controlling the dissolved oxygen rate of the fermentation system is to adjust the stirring speed and air volume. The stirring speed is 300~600rpm, and the air volume is 0.6~1.3m³ / h.

6. The method according to claim 1, characterized in that, The inoculation amount of the Corynebacterium glutamicum culture is 13%~15%.

7. The method according to any one of claims 1 to 6, characterized in that, The primary seed culture medium for culturing the *Corynebacterium glutamicum* bacterial suspension comprises the following components: glucose 60-70 g / L, magnesium sulfate 1-2 g / L, beet molasses 25-30 g / L, corn steep liquor 30-50 g / L, ferrous sulfate 0.04-0.06 g / L, manganese sulfate 0.02-0.05 g / L, potassium dihydrogen phosphate 3.5-5.5 g / L, copper sulfate 1-2 mg / L, zinc sulfate 0.6-0.8 mg / L, biotin 0.03-0.04 g / L, and a vitamin mixture 0.05-0.06 g / L.

8. The method according to claim 7, characterized in that, The initial dissolved oxygen rate of the primary seed culture medium is 100%; the culture temperature is 30~31℃; and the culture pressure is 0.05MPa.

9. The method according to claim 7, characterized in that, During the cultivation of the L-isoleucine-producing bacteria culture, the dissolved oxygen rate of the culture system is controlled at 30%~40%, and the pH value of the culture system is controlled at 7.2~7.4.

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