Method for shortening fermentation period of L-isoleucine

Abstract

The invention provides a method for shortening the fermentation period of L-isoleucine, and belongs to the technical field of amino acid fermentation. A method for shortening the fermentation period of L-isoleucine comprises the following steps: inoculating an L-isoleucine producing strain liquid into a fermentation culture medium for fermentation culture, and ending the fermentation culture when dissolved oxygen and pH value rise after a sugar solution in a fermentation system is exhausted. According to the L-isoleucine fermentation method, the nutritional ingredients are added at a time for L-isoleucine fermentation, feeding of sugar liquid and other nutritional ingredients in the fermentation period is omitted, the logarithmic phase of L-isoleucine production bacteria can be prolonged, thalli can be massively propagated, the period can be effectively shortened by 30%-40%, and meanwhile the yield and the conversion rate of L-isoleucine are guaranteed by adjusting the content of the nutritional ingredients.

Description

Technical Field

[0001] The invention belongs to the technical field of amino acid fermentation, and particularly relates to a method for shortening the fermentation cycle of L-isoleucine. Background Art

[0002] L-isoleucine, also known as isoleucine and 2-amino-3-methylvaleric acid, has a chemical formula of C6H 13 NO2 is one of the essential amino acids for the human body. L-isoleucine has important application value in the pharmaceutical, health care, food and other industries.

[0003] Currently, there are three main methods for producing L-isoleucine: extraction, chemical synthesis, and fermentation. Extraction and chemical synthesis methods are not suitable for industrial production due to difficulties in separating the produced L-isoleucine from its isomers, limited raw material sources, high production costs, and environmental pollution. Fermentation, on the other hand, offers mild conditions, environmental friendliness, and stable product quality, making it the most important method for producing L-isoleucine. However, existing fermentation methods for producing L-isoleucine generally have excessively long fermentation cycles, influenced by the fermentation process. Excessively long fermentation cycles can lead to increased production costs such as electricity, gas, and labor, and can also increase the probability of bacterial contamination during the fermentation process. Summary of the Invention

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

[0005] The present invention provides a method for shortening the L-isoleucine fermentation cycle, comprising the following steps: Corynebacterium glutamicum ( Corynebacterium glutamicum ) inoculating the bacterial liquid into the fermentation medium for fermentation culture, and terminating the fermentation culture when the dissolved oxygen and pH value in the fermentation system begin to rise; The fermentation medium includes the following components: 120-210 g / L of glucose, 2-3 g / L of ammonium sulfate, 0.25-1 g / L of magnesium sulfate, 15-20 g / L of beet molasses, 1.8-2.5 mg / L of ferrous sulfate, 2-3.5 mg / L of manganese sulfate, 0.55-1.0 g / L of potassium dihydrogen phosphate, 0.3-0.6 mg / L of copper sulfate, 0.4-0.7 mg / L of zinc sulfate, 6-10 mg / L of biotin, and 20-80 mg / L of a vitamin mixture.

[0006] Preferably, the fermentation medium comprises the following components: 125-167 g / L glucose, 2.2-2.8 g / L ammonium sulfate, 0.39-0.8 g / L magnesium sulfate, 17-19 g / L beet molasses, 1.95-2.15 mg / L ferrous sulfate, 2.5-3.5 mg / L manganese sulfate, 0.7-1.0 g / L potassium dihydrogen phosphate, 0.45-0.55 mg / L copper sulfate, 0.55-0.65 mg / L zinc sulfate, 7.5-8.5 mg / L biotin, and 60-70 mg / L vitamin mixture.

[0007] Preferably, the initial dissolved oxygen rate of the fermentation medium is 80% to 85%; the temperature of the fermentation culture is 31 to 33° C.; and the pressure of the fermentation culture is 0.03 to 0.15 MPa.

[0008] Preferably, during the fermentation culture period, the dissolved oxygen rate of the fermentation system is controlled to be 20% to 30% of the initial dissolved oxygen; and the pH value of the fermentation system is controlled to be 7.2 to 7.4.

[0009] Preferably, the method of 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.

[0010] Preferably, the inoculation amount of the Corynebacterium glutamicum bacterial solution is 13% to 15%.

[0011] Preferably, the primary seed culture medium for culturing the Corynebacterium glutamicum bacterial liquid comprises the following components: 60-70 g / L glucose, 1-2 g / L magnesium sulfate, 25-30 g / L beet molasses, 30-50 g / L corn steep liquor, 0.04-0.06 g / L ferrous sulfate, 0.02-0.05 g / L manganese sulfate, 3.5-5.5 g / L potassium dihydrogen phosphate, 1-2 mg / L copper sulfate, 0.6-0.8 mg / L zinc sulfate, 0.03-0.04 g / L biotin, and 0.05-0.06 g / L vitamin mixture; the culture temperature is 30-31 ° C.

[0012] 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°C.

[0013] Preferably, during the culture period of the L-isoleucine-producing bacteria liquid, the dissolved oxygen rate of the culture system is controlled to be 30% to 40%, and the pH value of the culture system is controlled to be 7.2 to 7.4.

[0014] Preferably, the Corynebacterium glutamicum includes the Corynebacterium glutamicum strain DXMC601; the deposit number of the Corynebacterium glutamicum strain DXMC601 is CCTCC NO: M 2025347.

[0015] The present invention provides a method for shortening the L-isoleucine fermentation cycle, comprising the following steps: Corynebacterium glutamicum ) the bacterial liquid is inoculated into a fermentation medium for fermentation culture, and fermentation culture is terminated when the sugar in the fermentation system is exhausted and the dissolved oxygen and pH value begin to rise; the fermentation medium comprises the following components: 120-210 g / L glucose, 2-3 g / L ammonium sulfate, 0.25-1 g / L magnesium sulfate, 15-20 g / L beet molasses, 1.8-2.5 mg / L ferrous sulfate, 2-3.5 mg / L manganese sulfate, 0.55-1.0 g / L potassium dihydrogen phosphate, 0.3-0.6 mg / L copper sulfate, 0.4-0.7 mg / L zinc sulfate, 6-10 mg / L biotin, and 20-80 mg / L vitamin mixture. The present invention addresses the problem of prolonged fermentation cycles caused by the addition of sugar solution and other nutrients during traditional fermentation to produce L-isoleucine. By creatively adding a base sugar and other nutrients to the fermentation medium at once, the logarithmic growth phase of the L-isoleucine-producing bacteria is significantly prolonged, allowing for massive bacterial growth and shortening the fermentation cycle by 30% to 40%. Furthermore, by optimizing the amounts of components added to the fermentation medium, the fermentation cycle is shortened while achieving a high L-isoleucine yield and maintaining a high conversion rate. The technical solution provided by the present invention can significantly shorten the fermentation cycle, reduce electrical usage and costs during the production process, and reduce the chance of bacterial contamination during the fermentation process, while also ensuring the yield and conversion rate of L-isoleucine.

[0016] Description of biological material deposit Corynebacterium glutamicum ( Corynebacterium glutamicum ) strain DXMC601, deposited in China Center for Type Culture Collection (abbreviated as CCTCC), located at Wuhan University, Wuhan, China, on March 3, 2025, with the deposit number CCTCC NO: M 2025347. DETAILED DESCRIPTION

[0017] The present invention provides a method for shortening the L-isoleucine fermentation cycle, comprising the following steps: Corynebacterium glutamicum ( Corynebacterium glutamicum ) The bacterial liquid is inoculated into the fermentation medium for fermentation culture, and the fermentation culture is terminated when the dissolved oxygen and pH value in the fermentation system begin to rise.

[0018] In the present invention, the problem of the traditional fed-batch method extending the fermentation cycle is addressed. The method is to form a fermentation medium by adding a substrate sugar and other nutrients at one time, omitting the fed-batch step, so that the L-isoleucine-producing bacteria can proliferate in large quantities in a short period of time. At the same time, the addition amount of the medium components is adjusted to meet the metabolic needs of the L-isoleucine-producing bacteria, thereby producing L-isoleucine in large quantities through fermentation, shortening the fermentation cycle while ensuring the yield and conversion rate of L-isoleucine.

[0019] In the present invention, the preparation method of the Corynebacterium glutamicum bacterial solution is preferably to inoculate Corynebacterium glutamicum in a primary culture medium for cultivation. In the embodiment of the present invention, the Corynebacterium glutamicum with the deposit number CCTCC NO: M 2025347 ( Corynebacterium glutamicum ) strain DXMC601 is an L-isoleucine-producing bacterium and is used to perform L-isoleucine fermentation to illustrate the technical effect of the present invention.

[0020] In the present invention, the primary seed culture medium preferably includes the following components: 60-70 g / L glucose, 2-3 g / L ammonium sulfate, 1-2 g / L magnesium sulfate, 25-30 g / L beet molasses, 30-50 g / L corn steep liquor, 0.04-0.06 g / L ferrous sulfate, 0.02-0.05 g / L manganese sulfate, 3.5-5.5 g / L potassium dihydrogen phosphate, 1-2 mg / L copper sulfate, 0.6-0.8 mg / L zinc sulfate, 0.03-0.04 g / L biotin, and 0.05-0.06 g / L vitamin mixture. The vitamin mixture preferably includes 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, and may also be 2:1:2. The preparation method of the primary seed culture medium is preferably to mix and dissolve glucose, magnesium sulfate and beet molasses and then sterilize them separately, dissolve the remaining components of the primary seed culture medium in a fermentation tank to a fixed volume for real sterilization, and combine the separately sterilized glucose, magnesium sulfate and beet molasses mixture with the remaining components. The sterilization conditions for the separate sterilization are preferably 118~121℃, 20~30min. The sterilization conditions for the real sterilization are preferably 121~122℃, 20~30min. The primary seed culture medium also preferably includes a defoaming agent. The concentration of the defoaming agent is preferably 0.08~0.1ml / L. The present invention has no special restrictions on the type of the defoaming agent, and the types of defoaming agents known in the art can be used. The culture is preferably carried out in a fermentation tank. The volume of the primary seed culture medium preferably contained in a 30L fermentation tank is preferably 13~14L.

[0021] In the present invention, the initial dissolved oxygen rate of the first-level seed culture medium is preferably 100%. The method for adjusting the initial dissolved oxygen rate of the first-level seed culture medium is preferably to adjust the initial dissolved oxygen rate of the first-level seed culture medium by stirring speed and ventilation volume, specifically, the stirring speed is preferably 600rpm, the tank pressure is 0.05mpa, the ventilation volume is 12L / min, and it is maintained stable for 5min. The culture conditions of the L-isoleucine production bacteria liquid are preferably: culture pressure 0.05mpa, controlling the dissolved oxygen rate of the culture system to 30%~40%, controlling the pH value of the culture system to 7.2~7.4, and the culture temperature to 30~31°C. When the dissolved oxygen rate of the culture system decreases below 30% during the culture process, the dissolved oxygen is adjusted to the above-mentioned appropriate range by increasing the stirring speed and ventilation volume.

[0022] In the present invention, the culturing is preferably performed 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.

[0023] In the present invention, before fermentation, the L-isoleucine producing bacteria liquid is inoculated into the fermentation medium. The inoculation amount of the L-isoleucine producing bacteria liquid is preferably 13% to 15%, and can be 14%.

[0024] In the present invention, the fermentation medium preferably includes the following components: 125-167 g / L glucose, 0.39-0.8 g / L magnesium sulfate, 17-19 g / L beet molasses, 1.95-2.15 mg / L ferrous sulfate, 2.5-3.5 mg / L manganese sulfate, 0.7-1.0 g / L potassium dihydrogen phosphate, 0.45-0.55 mg / L copper sulfate, 0.55-0.65 mg / L zinc sulfate, 7.5-8.5 mg / L biotin, and 60-70 mg / L vitamin mixture. The vitamin mixture preferably includes 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 preparation method of the fermentation medium is preferably to mix and dissolve glucose, magnesium sulfate and beet molasses and then sterilize them separately, while the remaining components in the fermentation medium are mixed and dissolved in a fermenter for actual sterilization. The sterilization conditions for the individual sterilization are preferably 118-121°C and 20-30 minutes. The actual sterilization conditions are preferably 121-122°C and 20-30 minutes. The fermentation medium is mixed with the separately sterilized glucose, magnesium sulfate and beet molasses and other components in other fermentation media before inoculation. The fermentation culture is preferably carried out in a fermenter. The volume of the fermentation medium contained in a 50L fermenter is preferably 24-25L.

[0025] In the present invention, before inoculation, the initial dissolved oxygen rate of the fermentation medium is 80% to 85%, and may be 81% to 84%, or 82% to 83%. The method for calibrating the dissolved oxygen in the fermentation medium is preferably to maintain a stable dissolved oxygen rate of 100% for 5 minutes at a temperature of 32°C, a pH of 7.4, a stirring speed of 600 rpm, a tank pressure of 0.05 MPa, and an air volume of 1 m³ / h. During the fermentation culture period, the fermentation temperature is preferably 31°C to 33°C, and may be 32°C; the pressure of the fermentation system is preferably 0.03 to 0.15 MPa, and may be 0.05 to 0.12 MPa, or 0.08 to 0.1 MPa; the stirring speed is preferably 300 to 600 rpm, and may be 400 to 500 rpm, or 450 rpm; and the air volume is preferably 0.6 to 1.3 m³ / L, and may be 0.6 to 1.2 m³ / L, or 0.7 to 0.8. As fermentation progresses, the dissolved oxygen in the fermentation system gradually decreases. When the dissolved oxygen rate falls below 20%, gradually increase the stirring rate and air volume to maintain the dissolved oxygen level between 20% and 30% while maintaining the tank pressure. If the dissolved oxygen level becomes too high in the later stages of fermentation, adjust the system by reducing the stirring rate while maintaining the air volume constant. Regularly monitor the OD value, residual sugar, ammonia nitrogen, and acid content of the culture liquid during the fermentation process. Fermentation is terminated after 10 minutes when the pH and dissolved oxygen levels reverse.

[0026] In the present invention, to further demonstrate the advantages of the inventive method, a fed-batch method was used to ferment L-isoleucine. Experiments showed that while achieving the same or similar L-isoleucine output and conversion rate as the present invention, the fermentation cycle required 36 hours, while the present invention only required 21 to 30 hours. Simultaneously, when the fed-batch method was used to ferment L-isoleucine, artificially controlling the fermentation time to 24 hours significantly reduced the output and conversion rate of L-isoleucine. Furthermore, Comparative Example 6, by improperly adjusting the components of the fermentation medium, shortened the fermentation cycle, but reduced the conversion rate of L-isoleucine. Therefore, this fermentation method is not conducive to reducing production costs.

[0027] The method for shortening the L-isoleucine fermentation cycle provided by the present invention is described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 A method for shortening the L-isoleucine fermentation cycle, comprising the following steps: Prepare the primary seed culture medium: Accurately weigh the following: 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 2:1:2 mass ratio), and 2ml of bubo. Dissolve these in water and bring the volume to 11L in a 30L fermenter. Sterilize the mixture at 121°C for 20 minutes, leaving a volume of 12.5L after sterilization. Weigh 950g glucose, 23g magnesium sulfate, and 400g beet molasses in water and dissolve them in water. Bring the volume to 1.5L in a fed-batch tank. Wrap the plug with gauze and kraft paper and sterilize in an autoclave at 121°C for 20 minutes. After sterilization, transfer the contents of the fed-batch tank to the seed tank using a peristaltic pump. Set the temperature to 30°C, pH to 7.4, stirring speed to 600 rpm, tank pressure to 0.05 MPa, and air volume to 12 L / min. Maintain stability for 5 minutes and then calibrate the dissolved oxygen to 100%. Corynebacterium glutamicum) strain DXMC601, deposit number CCTCC NO: M 2025347) was poured into the first-stage seed tank through the inoculation port. The agitation speed was set at 300 rpm, the air volume at 6 L / min, the tank pressure at 0.05 MPa, and the temperature was maintained constant. As the dissolved oxygen level decreased during fermentation, the rotational speed and air volume were increased to maintain a dissolved oxygen level of 30%-40%. After 8 hours of fermentation, the OD value was monitored every 2 hours for a total of 22 hours. The measured OD value was 0.653, and the pressure was maintained. Prepare the fermentation medium: Accurately weigh the following: 50 mg of ferrous sulfate, 70 mg of manganese sulfate, 20 g of potassium dihydrogen phosphate, 60 g of ammonium sulfate, 12 mg of copper sulfate, 14 mg of zinc sulfate, 0.15 g of biotin, 1.7 g of a vitamin mixture (VB1, VB3, and VB5 in a 3:1:3 mass ratio), and 2 ml of buprofen. Dissolve these in water and bring the volume to 16 L in a 50 L fermentor. Sterilize the mixture at 121°C for 20 min, resulting in an 18 L volume. Weigh 3 kg of glucose, 19 g of magnesium sulfate, and 430 g of beet molasses, dissolve them in water, and bring the volume to 6 L in a fed-batch tank. Wrap the plug with gauze and kraft paper and sterilize in an autoclave at 121°C for 20 min. After sterilization, transfer the contents of the fed-batch tank to the fermentor via a peristaltic pump. Set the temperature to 32°C, pH to 7.4, agitation to 600 rpm, tank pressure to 0.05 MPa, and air volume to 1 m³ / h. Maintain stability for 5 minutes, then calibrate the dissolved oxygen to 100%. Transfer the culture solution from the first-stage seed tank to the large tank via the transfer pipe, using a 4L transfer volume. Set the agitation to 300 rpm, tank pressure to 0.05 MPa, and air volume to 0.6 m³ / h to begin fermentation and tank control. Initially measure sugar at 12 g / dL during fermentation. As fermentation progresses, gradually increase the speed to maintain dissolved oxygen between 20% and 30% when the dissolved oxygen level drops below 20%. Once the speed reaches 350 rpm, increase the air volume by 0.8 m³ / h. Next, gradually increase the speed to 400 rpm, then increase the ventilation ratio to 1.0 m³ / h, and repeat this process until the highest conditions are achieved. In the later stages of fermentation, when dissolved oxygen levels rise, gradually reduce the speed while maintaining the same air volume until fermentation is complete. During the fermentation process, the pH value was adjusted by ammonia water. The OD value, residual sugar and ammonia nitrogen were tested every 2 hours. The acid content was tested starting at 12 hours. The OD detection method was a wavelength of 562nm and a dilution of 200 times. The residual sugar was detected using a biosensor, the ammonia nitrogen was detected using a titration method, and the acid content was detected using a liquid phase method. After a period of 21 hours, the pH rose. After 10 minutes of rising, the fermentation was removed from the tank. Example 2 The primary seed culture medium and the tank control method were the same as those in Example 1. The only difference between the fermentation culture medium and Example 1 was that the mass of glucose was changed to 4 kg, and the tank control method was the same.

[0029] Example 3 The primary seed culture medium and the tank control method were the same as those in Example 1. The only difference between the fermentation culture medium and Example 1 was that the mass of glucose was changed to 5 kg, and the tank control method was the same.

[0030] Example 4 The primary seed culture medium and the control tank method are consistent with those in Example 2, with the only difference being that the amount of magnesium sulfate in the fermentation medium is reduced by 10% to 17 g. The culture medium is dissolved with water, fixed to 16 L in a 50 L fermentor, and sterilized at 121 ° C for 20 min. The volume after sterilization is 18 L. 4 kg of glucose powder, 17 g of magnesium sulfate, and 430 g of beet molasses are weighed, dissolved in water, fixed to 6 L in a flow-addition tank, and the plug is wrapped with gauze and kraft paper and placed in an autoclave for sterilization at 121 ° C for 20 min. The control tank method remains consistent.

[0031] Example 5 The primary seed culture medium and the control tank method are consistent with those in Example 2, with the only difference being that the amount of magnesium sulfate in the fermentation medium is reduced by 20% to 15 g. The culture medium is dissolved with water, fixed to 16 L in a 50 L fermentor, and sterilized at 121 ° C for 20 min. The volume after sterilization is 18 L. Weigh 4 kg of glucose powder, 15 g of magnesium sulfate, and 430 g of beet molasses, dissolve them in water, and fix to 6 L in a flow-addition tank. Wrap the plug with gauze and kraft paper and sterilize it in an autoclave at 121 ° C for 20 min. The control tank method remains consistent.

[0032] Example 6 The seed tank culture medium and the control tank method are consistent with those in Example 2, with the only difference being that the amount of magnesium sulfate in the fermentation medium is reduced by 50% to 10 g. The culture medium is dissolved in water, fixed to 16 L in a 50 L fermentor, and sterilized at 121 ° C for 20 min. The volume after sterilization is 18 L. Weigh 4 kg of glucose powder, 10 g of magnesium sulfate, and 430 g of beet molasses, dissolve them in water, and fix the volume to 6 L in a fed-addition tank. Wrap the plug with gauze and kraft paper and sterilize it in an autoclave at 121 ° C for 20 min. The control tank method remains consistent.

[0033] Comparative Example 1 The culture medium and control method for the first seed tank were the same as those in Example 1. Instead of a single-dose feed of the fermentation tank base sugar, the sugar solution and the small material were fed continuously during the fermentation process. The fermentation medium components included: 8g potassium dihydrogen phosphate, 45g ammonium sulfate, 25mg ferrous sulfate, 40mg manganese sulfate, 10mg zinc sulfate, 10mg copper sulfate, 50mg biotin, and 1.7g vitamin mixture (VB1, VB3, and VB5 in a mass ratio of 3:1:3). After dissolving these materials, the volume was adjusted to 16L in a 50L fermenter. Sterilization was performed at 121°C for 20 minutes, resulting in a volume of 24L after sterilization. 1kg of glucose powder, 15g of magnesium sulfate, and 300g of beet molasses were weighed, dissolved in water, and brought to a volume of 1.5L in fed-batch tank 1. The plug was wrapped with gauze and kraft paper. Prepare a 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 feed tank 2. Wrap the plug with gauze and kraft paper. Prepare a small feed feed: culture medium containing 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 small feed in water and bring the volume to 3 L in feed tank 3. Wrap the plug with gauze and kraft paper. Sterilize feed tanks 1, 2, and 3 in an autoclave at 121°C for 20 min.

[0034] After sterilization, the material of the feed tank 1 is added into the tank at one time through a peristaltic pump. Set the temperature to 32°C, pH 7.4, stirring to 600r, tank pressure to 0.05mpa, and air volume to 1.3m³ / h. Maintain stability for 5 minutes and then calibrate the dissolved oxygen to 100%. Transplant the bacterial liquid in the first-level seed tank to the fermentation tank through the transplanting pipe, and the transplanting volume is 4L. Set the stirring to 250rpm, tank pressure to 0.05, and ventilation ratio to 0.3, and start fermentation control tank. After the pH rises, start the flow addition of sugar solution and small materials, and the flow addition ratio is sugar solution: small materials = 7:1 (mass ratio). The residual sugar is controlled at 0.2-0.3 throughout the process, and the dissolved oxygen control mode is the same as in Example 1. The fermentation cycle is 40h.

[0035] Comparative Example 2 The culture medium and fermentation conditions of Comparative Example 2 were the same as those of Comparative Example 1, except that Comparative Example 2 maintained a continuous feeding mode after the pH value returned to normal, but the fermentation period was 24 hours.

[0036] Comparative Example 3 The culture medium and fermentation conditions of Comparative Example 3 were the same as those of Comparative Example 1, except that the ratio of sugar solution to small material flow in Comparative Example 3 was changed to 5:1, and the tank was unloaded after a cycle of 36 hours.

[0037] Comparative Example 4 The culture medium and fermentation conditions of Comparative Example 4 were the same as those of Comparative Example 1, except that the ratio of sugar solution to small material flow in Comparative Example 4 was changed to 5:1, and the fermentation cycle was 24 hours.

[0038] Comparative Example 5 Comparative Example 5 used the same culture medium and fermentation conditions as Comparative Example 1, except that the glucose content in the sugar solution in Comparative Example 5 was increased by 50% (m / m). Specifically, 10 kg of powdered sugar was added to a volume of 18 L. The sugar solution to small batch feed ratio was changed to 5:1, and the fermentation was tanked after a 36-h fermentation period.

[0039] Comparative Example 6 The seed tank culture medium and the tank control method were the same as those in Example 2. The only difference between the fermentation medium and Example 1 was that 100 g of corn steep liquor was added to the fermentation medium, and the tank control method was the same.

[0040] Comparative Example 7 The seed tank culture medium and the control tank method are consistent with those in Example 2, with the only difference being that the amount of magnesium sulfate in the large tank culture medium is reduced by 70% to 5.7 g. The culture medium is dissolved in water, fixed to 16 L in a 50 L fermenter, and sterilized at 121 ° C for 20 min. The volume after sterilization is 18 L. Weigh 4 kg of glucose powder, 10 g of magnesium sulfate, and 430 g of beet molasses, dissolve them in water, and fix the volume to 6 L in a fed-addition tank. Wrap the plug with gauze and kraft paper and place it in an autoclave for sterilization at 121 ° C for 20 min. The control tank method remains consistent.

[0041] In Examples 1 to 6 and Comparative Examples 1 to 7, the determination methods of various indicators are as follows: The dissolved oxygen rate of the fermentation system was measured using a Mettler dissolved oxygen electrode, which showed 100% during the initial fermentation period; OD value: measured by visible spectrophotometer at a wavelength of 562 nm; pH value: measured with a METTER OLEDO benchtop pH meter; Residual reducing sugar content: measured using SBA-40C enzyme membrane analyzer; Ammonia nitrogen content: methyl red-methylene blue as indicator, titration with hydrochloric acid standard solution; L-isoleucine content: determined by liquid phase method; Sugar-acid conversion rate = L-isoleucine production / total sugar consumption × 100% Formula I.

[0042] L-isoleucine yield = acid content × lower tank volume Formula II; Among them, the total sugar consumption is the mass of glucose actually consumed.

[0043] The measurement results of Examples 1 to 6 and Comparative Examples 1 to 6 are shown in Table 1.

[0044] Table 1 Fermentation cycle, bacterial count, L-isoleucine production and conversion rate of different fermentation methods

[0045] The results of Examples 1-3 show that by switching to a one-time addition mode, the OD value increases rapidly, the cycle is shortened by 30%-40%, and the acid production and conversion rate are better than those in a continuous addition mode. However, the initial sugar content should not be too high.

[0046] It can be seen from Examples 4-6 that by using the one-time feeding mode, reducing the amount of magnesium sulfate in the fermentation medium to 20%, the cycle is extended by 3-4 hours, but the acid production and conversion rate are improved.

[0047] As can be seen from comparative examples 1, 3 and 5 results, using continuous flow-adding mode, different conditions are changed, acid production and conversion rate are lower than disposable addition mode, and the cycle is obviously longer. As can be seen from comparative examples 2 and 4 results, using continuous flow-adding mode, flow-adding fermentation to 24h, stopping fermentation tank, and lower filling acid content compared with Example 1 have significant decline, and conversion rate also has obvious reduction simultaneously. As can be seen from comparative example 6 result, in disposable material addition mode, nitrogen source nutrients are additionally added, and OD value rises fast, and fermentation cycle is further shortened, but conversion rate obviously reduces. As can be seen from comparative example 7 result, when reducing the concentration of magnesium sulfate to a certain concentration, L-isoleucine output and sugar acid conversion rate can be reduced.

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for shortening the L-isoleucine fermentation cycle, characterized in that: The following steps are involved: Corynebacterium glutamicum ( Corynebacterium glutamicum ) The bacterial liquid is inoculated into the fermentation medium for fermentation cultivation, and the fermentation is terminated when the sugar solution in the fermentation system is exhausted and the dissolved oxygen and pH value begin to rise; The fermentation medium includes the following components: 120-210 g / L of glucose, 2-3 g / L of ammonium sulfate, 0.25-1 g / L of magnesium sulfate, 15-20 g / L of beet molasses, 1.8-2.5 mg / L of ferrous sulfate, 2-3.5 mg / L of manganese sulfate, 0.55-1.0 g / L of potassium dihydrogen phosphate, 0.3-0.6 mg / L of copper sulfate, 0.4-0.7 mg / L of zinc sulfate, 6-10 mg / L of biotin, and 20-80 mg / L of a vitamin mixture.

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

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

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

4.

5. The method according to claim 4, characterized in that: The method of 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 bacterial liquid is 13% to 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 liquid comprises the following components: 60-70 g / L of glucose, 1-2 g / L of magnesium sulfate, 25-30 g / L of beet molasses, 30-50 g / L of corn steep liquor, 0.04-0.06 g / L of ferrous sulfate, 0.02-0.05 g / L of manganese sulfate, 3.5-5.5 g / L of potassium dihydrogen phosphate, 1-2 mg / L of copper sulfate, 0.6-0.8 mg / L of zinc sulfate, 0.03-0.04 g / L of biotin, and 0.05-0.06 g / L of a vitamin mixture.

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° C.; and the culture pressure is 0.05 MPa.

9. The method according to claim 7, characterized in that: During the culture period of the L-isoleucine production bacterial solution, the dissolved oxygen rate of the culture system is controlled to be 30% to 40%, and the pH value of the culture system is controlled to be 7.2 to 7.

4.

10. The method according to any one of claims 1 to 6 and claims 8 and 9, characterized in that: The Corynebacterium glutamicum is the Corynebacterium glutamicum strain DXMC601; The deposit number of the Corynebacterium glutamicum strain DXMC601 is CCTCC NO: M 2025347.

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