Fermentation enhancer and method for improving fermentation production efficiency of L-isoleucine

Abstract

The invention belongs to the technical field of amino acid preparation through microbial fermentation, and particularly relates to a fermentation enhancer and a method for improving the fermentation production efficiency of L-isoleucine. The invention provides a fermentation enhancer for improving the fermentation production efficiency of L-isoleucine, which can promote rapid growth and reproduction of thalli and metabolism of the thalli and improve the yield and the fermentation production efficiency of the L-isoleucine. The invention further provides a method for improving the fermentation production efficiency of the L-isoleucine by using the fermentation enhancer in the technical scheme, the fermentation process of the L-isoleucine is accurately controlled by feeding the glucose solution and feeding the nutrient solution containing the fermentation enhancer, so that thalli are quickly accumulated to produce the L-isoleucine, the fermentation period is shortened, and the production efficiency of the L-isoleucine is improved. The fermentation conversion rate is improved; meanwhile, L-isoleucine in fermentation liquor is rapidly crystallized and separated out of the fermentation liquor in the fermentation process, the feedback repression effect caused by accumulation of products is avoided, and the fermentation production efficiency of the L-isoleucine is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of preparing amino acids by microbial fermentation, and specifically relates to a fermentation enhancer and a method for improving the fermentation production efficiency of L-isoleucine. Background Art

[0002] L-isoleucine is also known as "isoleucine". Its chemical name is α-amino-β-methylvaleric acid, and its molecular formula is C6H 13 NO2, odorless, bitter, rhombus-shaped leaf-shaped or flaky crystals. L-isoleucine is a neutral aliphatic amino acid, a branched-chain amino acid, and one of the essential amino acids for the human body. It is widely used in medicine, food additives, and animal feed industries.

[0003] In the early days, L-isoleucine was mainly produced by chemical synthesis and protein hydrolysis. Due to the disadvantages of low yield and serious environmental pollution, it was gradually replaced by microbial fermentation. Microbial fermentation is a method of directly fermenting L-isoleucine using glucose as raw material by utilizing the biological metabolism of microorganisms. It has the advantages of low raw material cost, mild and easy to control reaction conditions, and low pollution.

[0004] The yield of L-isoleucine prepared by microbial fermentation in the prior art is low, and the utilization rate of fermentation production equipment is low, and the production efficiency is low. Therefore, it is necessary to propose a method that can improve the fermentation production efficiency of L-isoleucine and reduce the production cost. Summary of the invention

[0005] The object of the present invention is to provide a fermentation enhancer and method for improving the fermentation production efficiency of L-isoleucine, by adding the fermentation enhancer, accurately controlling the fermentation process, improving the metabolic activity of the bacteria, and separating the L-isoleucine in the fermentation broth from the fermentation broth in time, avoiding the accumulation of products causing feedback inhibition, and improving the L-isoleucine yield and fermentation production efficiency.

[0006] In order to solve the above technical problems, the following technical solutions are proposed: The invention provides a fermentation enhancer for improving the fermentation production efficiency of L-isoleucine. The fermentation enhancer uses water as a solvent and comprises components with the following concentrations: 18-20 g / L of hexadecyl sulfobetaine, 20-22 g / L of octadecyl dimethyl betaine, 0.3-0.5 g / L of chlorella growth factor, 2-4 g / L of cobalt chloride and 1-2 g / L of copper chloride.

[0007] The present invention provides a method for improving the fermentation production efficiency of L-isoleucine, comprising the following steps: Inoculating Corynebacterium glutamicum into a fermentation medium for fermentation culture, and when the glucose concentration in the fermentation broth is less than 0.5% (w / v), starting to flow-feed a glucose solution and a nutrient solution, wherein the nutrient solution contains the fermentation enhancer described in the above technical solution; during the fermentation culture, liquid ammonia is introduced to maintain the pH value of the fermentation culture at 7.3-7.5, and the volume of the introduced liquid ammonia is determined according to the change in the pH value; When the concentration of L-isoleucine in the fermentation broth reaches 4.3% to 4.6%, the L-isoleucine in the fermentation broth is separated, and the separated fermentation broth is continued to be fermented and cultured.

[0008] Preferably, the volume of the glucose solution added per unit time is determined according to the volume of liquid ammonia introduced per unit time, and the volume ratio of the liquid ammonia introduced to the glucose solution added per unit time is 1:(15~16); the volume of the nutrient solution added per unit time is determined according to the volume of the glucose solution added per unit time, and the volume ratio of the glucose solution added per unit time is (10~11):1.

[0009] Preferably, the nutrient solution uses water as a solvent and includes the following components in concentrations: 8-10 g / L of the fermentation enhancer described in the above technical solution, 0.6-0.8 g / L of betaine, 2-4 g / L of magnesium sulfate, 1-2 g / L of potassium dihydrogen phosphate, 13-15 mg / L of biotin, 15-18 mg / L of nicotinamide, 10-12 mg / L of calcium pantothenate, and 20-22 mg / L of thiamine.

[0010] Preferably, the fermentation temperature is 33-35°C, the fermentation pressure is 0.03-0.05 MPa, and the ventilation rate is 0.3-0.5 m3 / min per cubic meter of fermentation liquid. 3 Air.

[0011] Preferably, the fermentation medium uses water as a solvent and includes the following components in the following concentrations: yeast powder 5-8 g / L, glucose 10-15 g / L, magnesium sulfate 0.2-0.5 g / L, corn steep liquor 5-8 g / L, potassium dihydrogen phosphate 1-2 g / L, ammonium citrate 2-4 g / L, biotin 10-12 mg / L, nicotinamide 5-7 mg / L and calcium pantothenate 8-10 mg / L.

[0012] Preferably, the inoculation method of the Corynebacterium glutamicum includes seed liquid; the inoculation amount of the seed liquid is 20% to 25% of the volume of the fermentation medium.

[0013] Preferably, the concentration of the glucose solution is 50% to 55%.

[0014] Preferably, the number of separations of L-isoleucine in the fermentation broth during the fermentation culture is 2 to 3 times.

[0015] Preferably, the separation comprises pressing the fermentation fluid into a separation device.

[0016] Beneficial effects of the present invention: 1. The present invention provides a fermentation enhancer for improving the fermentation production efficiency of L-isoleucine. The hexadecyl sulfobetaine and octadecyl dimethyl betaine in the fermentation enhancer are quaternary ammonium surfactants, which can change the permeability of bacterial cell membranes, promote the transport of various nutrients into bacterial cells, and promote the release of L-isoleucine to the outside of cells. Chlorella growth factor (CGF) contains rich nucleoproteins, nucleic acids, ribonucleic acids, deoxyribonucleic acids, multiple vitamins, amino acids, polysaccharide components, complex protein bodies, enzymes, glycoproteins, etc., which can improve The metal ions cobalt chloride and copper chloride can not only assist CGF in being transported into bacterial cells through transport proteins, but also improve the metabolic enzyme activity of bacteria and promote the growth and reproduction of bacteria. Under the joint action of hexadecyl sulfobetaine and octadecyl dimethyl betaine surfactants, cobalt chloride and copper chloride metal ions and CGF nutrients, the cell membrane permeability of L-isoleucine fermentation bacteria is better, nutrients quickly enter the cells, the metabolic activity of bacteria is greatly enhanced, and the L-isoleucine fermentation production efficiency is improved.

[0017] 2. The present invention also provides a method for improving the fermentation production efficiency of L-isoleucine. The present invention adjusts the amount of liquid ammonia introduced by the change of pH, further feedback-adjusts the flow-feeding of glucose solution and the flow-feeding of nutrient solution containing the fermentation enhancer described in the above technical scheme, accurately controls the content of nutrients in the L-isoleucine fermentation process and the amount of synthetic raw materials for L-isoleucine, enables the bacteria to quickly accumulate and produce L-isoleucine, shortens the fermentation cycle, and improves the fermentation conversion rate.

[0018] 3. The present invention uses a separation device to quickly crystallize L-isoleucine in the fermentation broth at low temperature to separate the fermentation broth, and then uses nutrients and bacteria to continue fermentation culture, thereby avoiding feedback inhibition caused by product accumulation and L-isoleucine crystallization blocking the pipeline and forcing fermentation to stop, thereby achieving the effect of improving the fermentation yield and conversion rate, and ultimately improving the L-isoleucine fermentation production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the structure of an auxiliary device for improving the amino acid production efficiency used in the present invention; Figure 2 for Figure 1 Enlarged view of M in the middle; Figure 3 The figure is a schematic diagram of the installation position of the barrier component; Figure 4It is the structural schematic diagram of the coil; Figure 5 It is a structural schematic diagram of the supporting component; Figure 6 It is a structural schematic diagram of the spray component; Figures 1 to 6 middle: 1-tank body, 2-tee pipe, 21-feeding valve, 22-cleaning valve, 23-blocking component, 231-pipe cover, 232-movable blocking plate, 233-fixed blocking plate, 234-handle, 24-feeding pipeline, 25-cleaning pipeline, 26-tank body connecting pipe, 27-rubber layer, 3-coil, 31-coil inlet connecting pipe, 32-coil outlet connecting pipe, 33-coil connecting pipe, 4-support component, 41-fixed frame, 42-sliding frame, 43-support frame, 44-fixed plate, 5-spraying component, 6-tank body discharge pipe, 7-fermentation liquid outlet valve, 8-L-isoleucine outlet valve, 51-spraying port; Figure 7 Fermentation flow chart for improving the efficiency of L-isoleucine fermentation production. DETAILED DESCRIPTION

[0020] The present invention provides a fermentation enhancer for improving the fermentation production efficiency of L-isoleucine, comprising the following concentration components: Hexadecyl sulfobetaine 18~20g / L, octadecyl dimethyl betaine 20~22g / L, CGF 0.3~0.5g / L, cobalt chloride 2~4g / L and copper chloride 1~2g / L.

[0021] As an optional embodiment, the fermentation enhancer provided by the present invention includes 18-20 g / L of hexadecyl sulfobetaine, or 18.5-19.5 g / L. As an optional embodiment, the fermentation enhancer provided by the present invention includes 20-22 g / L of octadecyl dimethyl betaine, or 20.5-21.5 g / L. The hexadecyl sulfobetaine and octadecyl dimethyl betaine of the present invention can change the permeability of bacterial cell membranes, promote the transport of various nutrients such as CGF into bacterial cells, and promote the release of L-isoleucine outside the cells.

[0022] As an optional embodiment, the fermentation enhancer provided by the present invention may include CGF 0.3~0.5g / L, or 0.35~0.45g / L. CGF provides rich nutrients for bacterial cells, improves cell metabolic activity, and accelerates the conversion of glucose into L-isoleucine.

[0023] As an optional embodiment, the fermentation enhancer provided by the present invention includes 2-4 g / L of cobalt chloride, or 2.5-3.5 g / L. The fermentation enhancer provided by the present invention includes 1-2 g / L of copper chloride, or 1.2-1.6 g / L. The cobalt chloride and copper chloride of the present invention can not only assist CGF in being transported into bacterial cells through transport proteins, but also improve bacterial metabolic enzyme activity and promote bacterial growth and reproduction.

[0024] Under the joint action of the multi-components of hexadecyl sulfobetaine, octadecyl dimethyl betaine, CGF, cobalt chloride and cupric chloride, the fermentation enhancer provided by the invention improves the permeability of the cell membrane of L-isoleucine fermentation bacteria, enables nutrients to quickly enter the cells, greatly enhances the metabolic activity of the bacteria, and improves the L-isoleucine fermentation production efficiency.

[0025] As an optional embodiment, the fermentation enhancer of the present invention uses water as solvent.

[0026] The present invention has no particular limitation on the sources of the components in the fermentation enhancer, and sources familiar to those skilled in the art may be used.

[0027] The present invention provides a method for improving the fermentation production efficiency of L-isoleucine, comprising the following steps: Inoculating Corynebacterium glutamicum into a fermentation medium for fermentation culture, and when the glucose concentration in the fermentation broth is less than 0.5% (w / v), starting to flow-feed a glucose solution and a nutrient solution, wherein the nutrient solution contains the fermentation enhancer described in the above technical solution; during the fermentation culture, liquid ammonia is introduced to maintain the pH value of the fermentation culture at 7.3-7.5, and the volume of the introduced liquid ammonia is determined according to the change in the pH value; When the concentration of L-isoleucine in the fermentation broth reaches 4.3% to 4.6%, the L-isoleucine in the fermentation broth is separated, and the separated fermentation broth is continued to be fermented and cultured.

[0028] In the present invention, there is no particular limitation on the strain type of the Corynebacterium glutamicum, and any strain type of Corynebacterium glutamicum conventionally used for fermentation to prepare L-isoleucine in the art can be used.

[0029] As an optional embodiment, the present invention inoculates the seed liquid of Corynebacterium glutamicum into the fermentation medium for fermentation culture. The present invention has no special limitation on the preparation method of the seed liquid, and a conventional method can be used. The present invention has no special limitation on the method of inoculation, and a conventional method can be used. The inoculation amount of the seed liquid of the present invention can be 20%~25% of the volume of the fermentation medium, or it can be 24%~25%. As an embodiment, the fermentation medium of the present invention can use water as a solvent, and include the following components in concentrations: yeast powder 5~8g / L, glucose 10~15g / L, magnesium sulfate 0.2~0.5g / L, corn steep liquor 5~8g / L, potassium dihydrogen phosphate 1~2g / L, ammonium citrate 2~4g / L, biotin 10~12mg / L, nicotinamide 5~7mg / L and calcium pantothenate 8~10mg / L; in an embodiment of the present invention, the fermentation medium may include yeast powder 6 g / L, glucose 12g / L, magnesium sulfate 0.3g / L, corn steep liquor 5g / L, potassium dihydrogen phosphate 1g / L, ammonium citrate 3g / L, biotin 11mg / L, nicotinamide 5mg / L and calcium pantothenate 8mg / L; it may also include yeast powder 8g / L, glucose 15g / L, magnesium sulfate 0.2g / L, corn steep liquor 7g / L, potassium dihydrogen phosphate 1.5g / L, ammonium citrate 4g / L, biotin 12mg / L, nicotinamide 7mg / L and calcium pantothenate 10mg / L.

[0030] Usually, the optimal growth pH of microorganisms is neutral. However, since acidic substances are produced during their metabolism, the acidic pH of the fermentation liquid will affect the growth and reproduction of the microorganisms. Therefore, liquid ammonia is usually required to maintain the pH value at a neutral level during the microbial fermentation process. In the fermentation and culture process described in the present invention, liquid ammonia is introduced to adjust the pH value of the fermentation liquid to 7.3~7.5, or 7.4. As an optional embodiment, the temperature of the fermentation and culture described in the present invention is 33~35°C, or 34~35°C. The pressure of the fermentation and culture described in the present invention is 0.03~0.05MPa, or 0.05MPa. As an optional embodiment, the fermentation and culture described in the present invention is carried out under ventilation conditions, and the ventilation volume can be 0.3~0.5m 3 Air / m 3 Fermentation liquid / min, can also be 0.5m 3 Air / m 3 Fermentation liquid / min.

[0031] The present invention maintains a pH value of 7.3 to 7.5 during the fermentation culture, and adjusts the pH value of the fermentation culture to 7.3 to 7.5 by introducing liquid ammonia. The pH value conditions of the fermentation culture maintained by the present invention are conducive to the growth and reproduction of Corynebacterium glutamicum to produce L-isoleucine, preferably liquid ammonia is introduced from the beginning, and the liquid ammonia rate is determined according to the change of pH to ensure that the pH value of the system is maintained within the range of 7.3 to 7.5.

[0032] After the fermentation culture begins, the present invention preferably monitors the glucose mass concentration in the fermentation broth. When the glucose mass concentration in the fermentation broth of the present invention is less than 0.5% (w / v) for the first time, the glucose solution and the nutrient solution are started to be added. The present invention preferably continues to add the glucose and nutrient solution after the start of the addition. In the present invention, the nutrient solution contains the fermentation enhancer described in the above technical solution. Glucose is a substrate for Corynebacterium glutamicum to produce L-isoleucine by fermentation, and the nutrient solution provides nutrients for the growth and reproduction of Corynebacterium glutamicum.

[0033] The speed of the flow-added glucose solution of the present invention is adjusted according to the feedback of the change of pH, that is, it is determined according to the speed of the liquid ammonia introduced, and the speed of the flow-added nutrient solution is determined according to the speed of the flow-added glucose solution. In the present invention, the pH value of the fermentation culture is adjusted to 7.3~7.5 by introducing liquid ammonia, and the flow acceleration of the liquid ammonia is controlled by a flowmeter according to the feedback of the change of the pH value of the fermentation liquid. The flow acceleration of the glucose solution and the liquid ammonia solution of the present invention are also interlocked with the flow rate by a flowmeter. The volume of the flow-added glucose solution per unit time is determined according to the volume of the liquid ammonia introduced per unit time, and the ratio of the flow rate of the liquid ammonia to the flow rate of glucose is 1:15~16; that is, the volume ratio of the liquid ammonia of the present invention to the flow-added glucose solution per unit time is 1:(15~16); preferably 1:15.5. For example, in the embodiment, when the instantaneous flow rate of the liquid ammonia is 1m 3 / h, then the instantaneous flow rate of glucose is 15~16m 3 / h. In the present invention, the glucose solution is added simultaneously with the nutrient solution, and the flow acceleration of the glucose solution and the nutrient solution is also interlocked with the flow rate by using a flow meter. For example, the instantaneous flow rate of glucose is 10-11m / s. 3 / h, the instantaneous flow rate of the nutrient solution is 1m 3 / h. As an optional embodiment, the present invention determines the volume of the nutrient solution added per unit time according to the volume of the glucose solution added per unit time, and the volume ratio of the glucose solution to the nutrient solution per unit time is (10-11):1, or 10.5:1. As an optional embodiment, the concentration of the glucose solution added per unit time of the present invention is 50%-55% (w / v), or 53%-55% (w / v).

[0034] The change of pH during the fermentation process of the present invention, that is, the frequency of liquid ammonia introduction, directly reflects the growth rate of the microorganism. At the beginning of fermentation, the microorganism grows slowly, the metabolic rate is low, and the acidic substances produced in the metabolic process are also less. The pH adjustment rate is slow, that is, the frequency of liquid ammonia introduction is relatively low; as the fermentation cycle is extended, the microorganism grows vigorously, the metabolic rate increases, and the acidic substances produced in the metabolic process also increase. The pH adjustment rate is accelerated, that is, the frequency of liquid ammonia introduction increases. The sugar flow acceleration is adjusted by feedback of pH changes to accurately control the L-isoleucine fermentation process, and the fermentation enhancer is added at the same time to provide nutrients for microbial reproduction and metabolism, so that the bacterial metabolism is more conducive to the accumulation and production of L-isoleucine, shorten the fermentation cycle, and improve the fermentation conversion rate.

[0035] In the present invention, when the glucose concentration in the fermentation broth is less than 0.5% (w / v), the glucose solution and the nutrient solution are started to be fed, wherein the nutrient solution contains the fermentation enhancer described in the above technical solution.

[0036] As an optional embodiment, the nutrient solution uses water as a solvent and includes the following components in concentrations: 8-10 g / L of the fermentation enhancer described in the above technical solution, 0.6-0.8 g / L of betaine, 2-4 g / L of magnesium sulfate, 1-2 g / L of potassium dihydrogen phosphate, 13-15 mg / L of biotin, 15-18 mg / L of nicotinamide, 10-12 mg / L of calcium pantothenate, and 20-22 mg / L of thiamine.

[0037] As an optional embodiment, the nutrient solution of the present invention uses water as a solvent. The components of the nutrient solution provided by the present invention include betaine 0.6~0.8g / L, or 0.65~0.75g / L. The components of the nutrient solution provided by the present invention include magnesium sulfate 2~4g / L, or 2.5~3.5g / L. The components of the nutrient solution provided by the present invention include potassium dihydrogen phosphate 1~2g / L, or 1.3~1.8g / L. The components of the nutrient solution provided by the present invention include biotin 13~15mg / L, or 13.5~14.5mg / L. The components of the nutrient solution provided by the present invention include nicotinamide 15~18mg / L, or 15.5~17.5mg / L. The components of the nutrient solution provided by the present invention include calcium pantothenate 10~12mg / L, or 10.5~11.5mg / L. The components of the nutrient solution provided by the present invention include 20-22 mg / L of thiamine, or 20.5-21.5 mg / L.

[0038] Generally, the solubility of L-isoleucine is low. As the fermentation culture proceeds, the concentration of L-isoleucine in the fermentation broth gradually increases. When the content in the fermentation broth is ≥4.5%, crystallization begins to occur. If the crystallization is serious, it is easy to block the fermentation tank and affect the fermentation production, and the accumulation of products during the fermentation process often causes feedback inhibition. Generally, when the fermentation reaches the L-isoleucine content of 4.0%~4.5%, the fermentation ends. At this time, the tank is stopped, and not only the yield is low, but also the utilization rate of the fermentation production equipment is low, and the production efficiency is low. Therefore, when the concentration of L-isoleucine in the fermentation broth reaches 4.3%~4.6%, the L-isoleucine in the fermentation broth is separated, and the fermentation broth after separation continues to be fermented and cultured. The separation method of the present invention can include forcing the fermentation liquid into a separation device. The separation device can make the L-isoleucine in the fermentation broth crystallize rapidly under low temperature, quickly separate the fermentation broth, and the bacteria and nutrients in the fermentation broth after separation are returned to the fermentation equipment to continue fermentation and culture, so as to achieve the purpose of improving the yield of L-isoleucine and the fermentation production efficiency. The separation device described in the present invention may include a fermentation auxiliary device, which is arranged in a tank body. The auxiliary device includes a plurality of coils arranged at equal intervals from top to bottom in the tank body, adjacent coils are connected to each other, and adjacent layers of coils are arranged alternately; a spray component is arranged in the tank body, and the spray component is located above the coil; the spray component is in a cross structure, and a plurality of spray ports are arranged at the bottom of the spray component.

[0039] As an optional implementation, see Figure 1 and Figure 3 , the auxiliary device for improving the amino acid production efficiency provided by the present invention is arranged in the tank body 1. The auxiliary device includes a plurality of coils 3 arranged at equal intervals from top to bottom in the tank body 1, adjacent coils 3 are connected to each other, two adjacent layers of coils 3 are staggered, and the plurality of coils 3 are connected by a coil connecting pipe 33; the coil 3 at the bottom is connected to a coil inlet connecting pipe 31, one end of which passes through the side wall of the tank body 1; the coil 3 at the top is connected to a coil outlet connecting pipe 32, one end of which passes through the side wall of the tank body 1. Preferably, the coil inlet connecting pipe 31 is located on the opposite side of the coil outlet connecting pipe 32.

[0040] As an optional embodiment, the coil 3 is fixed in the tank body 1 by a support component 4, and a hollow structure is provided inside the support component 4. The coil connection pipe 33 between two adjacent layers of coils 3 can pass through the hollow structure of the support component 4 to connect. This arrangement not only reduces the weight of the support component 4, but also makes the connection between two adjacent layers of coils 3 smoother and more convenient. In order to adapt to the structure of the staggered arrangement of two adjacent layers of coils 3, the support component 4 is set to a retractable structure. See the structure for details. Figure 5Specifically, the support component 4 includes a fixed frame 41, a sliding frame 42 is sleeved in the fixed frame 41, and a support frame 43 is connected to the end of the sliding frame 42. The sliding distance of the sliding frame 42 can be adjusted according to the needs of the coil 3 to make the support frame 43 support the coil 3 more stably. A fixing plate 44 is also provided on the support component 4. The fixing plate 44 is an arc-shaped structure, and its curvature is adapted to the curvature of the side wall of the tank body 1. The fixing plate 44 can be connected to the tank body 1 by bolts, and can also be pasted on the tank body 1 using existing pasting technology.

[0041] The coil 3 can be fixed on the inner wall of the tank body 1 through the supporting component 4 to prevent the coil 3 from shaking and causing danger during operation.

[0042] As an optional embodiment, a detachable blocking member 23 is provided at the center of the three-way pipe 2, see Figures 1 to 3 The blocking component 23 includes a pipe cover 231, and a handle 234 is provided on the top of the pipe cover 231. The pipe cover 231 is connected to a fixed blocking plate 233, and the height of the fixed blocking plate 233 is 1 / 2 of the diameter of the three-way pipe 2.

[0043] As an optional implementation, see Figure 1 and Figure 6 The tank body 1 provided by the present invention is provided with a spraying component 5, and the spraying component 5 is located above the coil 3. The spraying component 5 is in a cross structure, and a plurality of spraying ports 51 are arranged at the bottom of the spraying component 5. By arranging the spraying component in a cross structure, the fermentation liquid containing amino acids can be sprayed more evenly in the entire tank body 1.

[0044] As an optional embodiment, the top of the spray component 5 is connected to the third pipe opening of the three-way pipe 2, see Figures 1 to 3, the other two pipe openings of the three-way pipe 2 are located outside the tank body 1 and are respectively provided with a feed valve 21 and a cleaning valve 22; a detachable barrier component 23 is provided at the center of the three-way pipe 2, and the barrier component 23 includes a pipe cover 231, a handle 234 is provided on the top of the pipe cover 231, and the pipe cover 231 is connected to a fixed barrier plate 233, and a movable barrier plate 232 is hinged at the bottom of the fixed barrier plate 233, and the movable barrier plate 232 extends into the third pipe opening of the three-way pipe 2. The height of the fixed barrier plate 233 is 1 / 2 of the diameter of the three-way pipe 2. A corrosion-resistant rubber layer 27 is provided at the third pipe opening of the three-way pipe 2. In order to prevent the movable barrier plate 232 from swinging left and right to cause collision damage to the three-way pipe 2, a corrosion-resistant rubber layer 27 is provided at the third pipe opening of the three-way pipe 2. The pipe opening of the feed valve 21 provided on the three-way pipe 2 is connected to the feed pipe 24, the pipe opening of the cleaning valve 22 provided on the three-way pipe 2 is connected to the cleaning pipe 25, and the third pipe opening of the three-way pipe 2 is connected to the tank body connecting pipe 26. The bottom of the tank body 1 is connected to the tank body discharge pipe 6, and the tank body discharge pipe 6 is connected to the fermentation liquid outlet pipe and the amino acid outlet pipe, the fermentation liquid outlet pipe is provided with a fermentation liquid outlet valve 7, and the amino acid outlet pipe is provided with an L-isoleucine outlet valve 8. A sight glass is provided on the tank body 1. The present invention uses a fermentation production auxiliary device to quickly crystallize L-isoleucine in the fermentation liquid and separate the fermentation liquid, so that the concentration of L-isoleucine in the fermentation liquid is reduced, and nutrients and bacteria enter the fermentation tank again to continue fermentation and cultivation, thereby avoiding the feedback inhibition caused by the accumulation of products, and L-isoleucine crystals blocking the pipeline and forcing the fermentation to stop, thereby achieving the purpose of increasing the fermentation yield, improving the utilization rate of the fermentation tank and improving the production efficiency.

[0045] As an optional embodiment, the present invention inoculates Corynebacterium glutamicum into a fermentation medium for fermentation culture. When the concentration of L-isoleucine in the obtained fermentation liquid reaches 4.3% to 4.6% for the first time, L-isoleucine in the fermentation liquid is separated for the first time, and the separated fermentation liquid is returned to the fermentation equipment for continued fermentation culture. The fermentation culture is still carried out according to the temperature, ventilation volume, and pressure described in the above technical scheme. At the same time, during the fermentation culture, glucose solution, nutrient solution, and liquid ammonia are added according to the above technical scheme. When the concentration of L-isoleucine in the fermented liquid reaches 4.3% to 4.6% for the second time, L-isoleucine in the fermentation liquid is separated for the second time, and the separated fermentation liquid is returned to the fermentation equipment for continued fermentation culture. When the concentration of L-isoleucine in the fermented liquid reaches 4.3% to 4.6% for the third time, the fermentation culture ends. During the second and third fermentation cultures of the present invention, no fermentation medium is added, and only glucose solution and nutrient solution are added. The components in the nutrient solution can provide nutrients for the growth of the bacteria and synthesize L-isoleucine. During the fermentation culture process of the present invention, L-isoleucine can be separated from the fermentation broth 2 to 3 times. If the separation is continued for the fourth time or more, the bacterial cell activity declines significantly, and the conversion rate and fermentation production intensity are greatly reduced.

[0046] The present invention provides a method for improving the fermentation production efficiency of L-isoleucine by using the nutrient solution described in the above technical solution. The present invention adjusts the flow acceleration of the glucose solution by feedback of the change of pH, and feeds the nutrient solution containing the fermentation enhancer, accurately controls the fermentation process of L-isoleucine, makes the bacteria accumulate quickly to produce L-isoleucine, shortens the fermentation cycle, and improves the fermentation conversion rate. At the same time, the fermentation production auxiliary device is used to quickly crystallize the L-isoleucine in the fermentation liquid and separate the fermentation liquid, and the nutrients and bacteria enter the fermentation tank again to continue fermentation and cultivation, thereby achieving the purpose of increasing the fermentation yield, improving the utilization rate of the fermentation tank and the production efficiency.

[0047] 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.

[0048] The present invention does not specifically limit the strain type of Corynebacterium glutamicum used in the following examples and comparative examples, and any strain type conventionally used in the art for fermentation to prepare L-isoleucine can be used. The present invention does not specifically limit the sources of the culture medium components and detection reagents used, and they can be purchased through conventional commercial channels. The total volume of the fermentation tank used in the present invention is 75m 3 .

[0049] The method for detecting the L-isoleucine content in the following examples and comparative examples is paper chromatography, and the method is as follows: Dilute the fermentation broth before measuring. Dilute the sample according to the content of L-isoleucine in the fermentation broth. The amino acid content in the dilution is about 2.0%. Use a spotting needle to spot the dilution on a 30*25cm filter paper. The spotting line is 1.5cm away from the bottom of the filter paper, with a spacing of 2.0cm. The spotting volume is 1μL. At the same time, spot a standard product with an amino acid concentration of 2.0%.

[0050] Place the spotted filter paper in a chromatography cylinder pre-filled with a developing agent, develop it by the ascending method at 25°C, and take it out to dry when the front edge of the developing agent reaches 1 cm above the upper end of the filter paper. Fold and unfold the dried filter paper, spray it with a color developer, heat it in an oven for color development, and dry it at 105°C for 5 minutes until purple-red amino acid spots appear on the filter paper. Cut off the purple-red spots on the filter paper, soak them in 5mL of eluent for 40 minutes (cover the mouth of the test tube to prevent ethanol from volatilizing), and measure the absorbance at 506nm. Use the absorbance value measured by the standard product to make a standard curve, and then check the L-isoleucine content of the measured dilution on the standard curve, and then multiply the L-isoleucine content of the dilution by the dilution multiple to obtain the L-isoleucine content in the fermentation broth sample. Among them, the developing agent is n-butanol, glacial acetic acid and distilled water, and the volume ratio of n-butanol, glacial acetic acid and distilled water is 36:9:15. The color developer is a ninhydrin acetone solution with a mass concentration of 0.5%. The eluent is a mixture of copper sulfate pentahydrate solution and ethanol, the mass concentration of the copper sulfate pentahydrate solution is 0.2%, the volume concentration of ethanol is 75%, and the volume ratio of the copper sulfate pentahydrate solution to ethanol is 2:38.

[0051] The same strain of Corynebacterium glutamicum was used in the following examples and comparative examples of the present invention.

[0052] The Corynebacterium glutamicum seed solution involved in the following examples and comparative examples was diluted 50 times with water, and the dilution was measured for absorbance OD at a wavelength of 600 nm by a spectrophotometer. 600 , when OD 600 When the OD value of the Corynebacterium glutamicum seed solution reaches 0.6-0.8, the Corynebacterium glutamicum seed solution is inoculated into the fermentation medium; the OD value of the Corynebacterium glutamicum seed solution used in different embodiments and comparative examples is 600 same.

[0053] The fermentation flow chart for improving the fermentation efficiency of L-isoleucine is shown in Figure 7 .

[0054] Example 1 A method for improving the fermentation production efficiency of L-isoleucine, comprising the following steps: 1. Inoculate the fermentation medium with a Corynebacterium glutamicum seed solution at a volume ratio of 24% for fermentation culture. The fermentation culture consists of a first fermentation, a second fermentation and a third fermentation, and the first fermentation, the second fermentation and the third fermentation are carried out in sequence.

[0055] (1) First fermentation culture Parameters: The fermentation temperature is controlled at 35°C, and the fermentation tank pressure is controlled at 0.05MPa. The fermentation is carried out under ventilation conditions, with a ventilation rate of 0.5m3 / min per cubic meter of fermentation liquid. 3 Air (i.e. ventilation volume 0.5m 3 Air / m 3 The flow rate of liquid ammonia is measured by a flow meter, and the pH value of the entire fermentation process is adjusted to 7.4 by introducing liquid ammonia, that is, the flow rate of liquid ammonia is confirmed based on the feedback of the change in the pH value of the fermentation liquid. At the same time, the glucose concentration in the fermentation liquid is monitored in real time. When the glucose concentration in the fermentation liquid is first detected to be less than 0.5% (w / v), glucose solution and nutrient solution are added to continue the entire fermentation process. The flow meter flow rate of liquid ammonia is interlocked with the flow meter flow rate of glucose solution. For example, the instantaneous flow rate of liquid ammonia is 0.1m 3 / h, the instantaneous flow rate of glucose is 1.5m 3 / h, that is, the volume ratio of liquid ammonia solution to glucose solution is controlled to be 1:15 per unit time. The nutrient solution is added at the same time as the glucose solution, and the flow meter flow rate of the glucose solution is interlocked with the flow meter flow rate of the nutrient solution. For example, the instantaneous flow rate of glucose is 1.5m 3 / h, the instantaneous flow rate of the nutrient solution is 0.15m 3 / h, that is, the volume ratio of the glucose solution to the nutrient solution per unit time is controlled to be 10:1. The concentration of the glucose solution to be fed is 55% (w / v).

[0056] Separation: When the L-isoleucine content in the fermentation liquid obtained by the first fermentation culture is 4.3%, the specific process of pressing the fermentation liquid into the fermentation production auxiliary device through the pressure difference is: open the feed valve 21 on the feed pipe 24, and at the same time open the fermentation liquid outlet valve 7, then close the cleaning valve 22, and pass the fermentation liquid containing L-isoleucine through the feed pipe 24. Under the action of the movable baffle plate 232, the fermentation liquid containing L-isoleucine is smoothly passed into the tank body 1, and the movable baffle plate 232 can prevent the fermentation liquid containing L-isoleucine from entering the cleaning pipe 25.

[0057] The fermentation liquid containing L-isoleucine is uniformly sprayed on the coils 3 of each layer through the multiple spray ports 51 on the spray component 5. At this time, cold water is introduced into the coils 3, and L-isoleucine is rapidly condensed on the coils 3 after being cooled, thereby realizing rapid separation of L-isoleucine from the fermentation liquid. The coils 3 of two adjacent layers are arranged in a staggered manner, so that the coils 3 can contact the fermentation liquid containing amino acids over a larger area, greatly increasing the amount of amino acids condensed on the coils 3 when being cooled. When the L-isoleucine in the fermentation liquid passes through the coils with a lower temperature, it rapidly crystallizes and adheres to the coils. After condensation, the nutrients and bacteria in the remaining fermentation liquid pass through the fermentation liquid outlet pipe connected to the tank body discharge pipe 6, and the fermentation liquid outlet valve 7 is opened, and enters the fermentation tank again to continue the second fermentation. The L-isoleucine outlet pipe is provided with an L-isoleucine outlet valve 8.

[0058] After the feeding is completed, the feeding valve 21 and the fermentation liquid outlet valve 7 are closed, hot water or steam is introduced into the coil 3, the temperature of the coil 3 is increased, and the L-isoleucine attached to the coil 3 is dissolved. The cleaning valve 22 is opened, a small amount of clean hot water is introduced for flushing, and then the L-isoleucine outlet valve 8 is opened to discharge the dissolved L-isoleucine solution out of the tank body 1, and then enter the subsequent production link.

[0059] At the end of production, alkaline solution or acid solution may be introduced through the cleaning valve 22 to clean the tank body 1 and the coil 3 .

[0060] In order to facilitate viewing of the situation in the tank body 1 , a sight glass may be provided on the tank body 1 , and a manhole may also be provided for operators to inspect and clean the crystals remaining in the tank body.

[0061] (2) Second fermentation culture: The parameters are the same as those of the first fermentation culture.

[0062] Separation: When the L-isoleucine content in the fermentation broth obtained from the second fermentation culture reaches 4.3%, the L-isoleucine is rapidly separated again by the fermentation production auxiliary device. The separation parameters are the same as those in step (1). The nutrients and bacteria in the fermentation broth continue to undergo a third fermentation.

[0063] (3) The third fermentation culture: the parameters are the same as those of the first fermentation culture.

[0064] When the L-isoleucine content in the fermentation liquid obtained by the third fermentation reaches 4.3%, the fermentation culture ends. The fermentation indexes are shown in Table 1.

[0065] The fermentation medium uses water as solvent, and the component concentrations are as follows: yeast powder 6g / L, glucose 12g / L, magnesium sulfate 0.3g / L, corn steep liquor 5g / L, potassium dihydrogen phosphate 1g / L, ammonium citrate 3g / L, biotin 11mg / L, nicotinamide 5mg / L and calcium pantothenate 8mg / L.

[0066] The nutrient solution uses water as solvent, and the component concentrations are as follows: betaine 0.7g / L, magnesium sulfate 2g / L, potassium dihydrogen phosphate 1g / L, biotin 13mg / L, nicotinamide 16mg / L, calcium pantothenate 11mg / L, thiamine 20mg / L and fermentation enhancer 8g / L; the fermentation enhancer uses water as solvent, and the component concentrations are as follows: hexadecyl sulfobetaine 18g / L, octadecyl dimethyl betaine 20g / L, CGF 0.3g / L, cobalt chloride 2g / L, copper chloride 1g / L.

[0067] Example 2 A method for improving the fermentation production efficiency of L-isoleucine, comprising the following steps: The Corynebacterium glutamicum seed solution is inoculated into the fermentation medium at a volume ratio of 25% for fermentation culture. The fermentation culture consists of a first fermentation, a second fermentation and a third fermentation, and the first fermentation, the second fermentation and the third fermentation are carried out in sequence.

[0068] (1) First fermentation culture: Parameters: The fermentation temperature is controlled at 34°C, the pressure is 0.04MPa, and the ventilation volume is 0.4m3 / min per cubic meter of fermentation liquid. 3 Air (i.e. ventilation volume 0.4m 3 Air / m 3 The flow rate of liquid ammonia is measured by a flow meter, and the pH value of the entire fermentation process is adjusted to 7.3 by adding liquid ammonia, that is, the flow rate of liquid ammonia is confirmed based on the feedback of the change in the pH value of the fermentation liquid. At the same time, the glucose concentration in the fermentation liquid is monitored in real time. When the glucose concentration in the fermentation liquid is first detected to be less than 0.5% (w / v), glucose solution and nutrient solution are added to continue the entire fermentation process. The flow meter flow rate of liquid ammonia is interlocked with the flow meter flow rate of glucose solution. The instantaneous flow rate of liquid ammonia is 1m 3 / h, the instantaneous flow rate of glucose is 16m 3 / h. That is, the volume ratio of liquid ammonia solution to glucose solution is controlled to be 1:16 per unit time. The nutrient solution is added at the same time as the glucose solution. The flow meter flow rate of the glucose solution is interlocked with the flow meter flow rate of the nutrient solution. The instantaneous flow rate of the nutrient solution is 1m 3 / h, the instantaneous flow rate of glucose is 11m 3 / h, that is, the volume ratio of the glucose solution to the nutrient solution per unit time is controlled to be 11:1. The concentration of the glucose solution is 53% (w / v).

[0069] Separation: When the L-isoleucine content in the fermentation broth obtained from the first fermentation is 4.4%, the fermentation broth is pressed into the fermentation production auxiliary device through a pressure difference. The specific process of pressing the fermentation broth into the fermentation production auxiliary device through a pressure difference is the same as the separation in step (1) of Example 1.

[0070] (2) Second fermentation culture: Parameters: Same as those for the first fermentation.

[0071] Separation: When the L-isoleucine content in the fermentation broth obtained from the second fermentation culture reaches 4.3%, the L-isoleucine is rapidly separated again by the fermentation production auxiliary device. The separation parameters are the same as those in step (1) of Example 1. The nutrients and bacteria in the fermentation broth continue to undergo a third fermentation.

[0072] (3) The third fermentation culture: Parameters: Same as those for the first fermentation.

[0073] Separation: When the L-isoleucine content in the fermentation liquid obtained from the third fermentation reaches 4.4%, the fermentation ends. The fermentation indexes are shown in Table 1.

[0074] The fermentation medium uses water as solvent, and the component concentrations are as follows: yeast powder 8g / L, glucose 15g / L, magnesium sulfate 0.2g / L, corn steep liquor 7g / L, potassium dihydrogen phosphate 1.5g / L, ammonium citrate 4g / L, biotin 12mg / L, nicotinamide 7mg / L and calcium pantothenate 10mg / L.

[0075] The nutrient solution uses water as solvent, and the component concentrations are as follows: betaine 0.8g / L, magnesium sulfate 3g / L, potassium dihydrogen phosphate 2g / L, biotin 14mg / L, nicotinamide 17mg / L, calcium pantothenate 12mg / L, thiamine 21mg / L and fermentation enhancer 10g / L; the fermentation enhancer uses water as solvent, and the component concentrations are as follows: hexadecyl sulfobetaine 20g / L, octadecyl dimethyl betaine 22g / L, CGF 0.5g / L, cobalt chloride 4g / L, copper chloride 2g / L.

[0076] Comparative Example 1 Same as Example 1, the only difference is that the flow rate of the glucose solution is a uniform flow rate, and the flow rate is selected to be 0.9m 3 / h, other conditions remain unchanged. Fermentation indicators are shown in Table 1.

[0077] Comparative Example 2 The same as Example 1, the only difference is that the flow rate of the nutrient solution is a uniform flow rate, and the flow rate is selected to be 0.1m 3 / h, other conditions remain unchanged. Fermentation indicators are shown in Table 1.

[0078] Comparative Example 3 The same as Example 1, the only difference is that the nutrient solution fed does not contain a fermentation enhancer. The fermentation indexes are shown in Table 1.

[0079] Comparative Example 4 The same as Example 1, the only difference is that the fermentation liquid does not enter the fermentation auxiliary device during the fermentation, and the fermentation ends when the content of L-isoleucine in the fermentation liquid is ≥4.3%. The fermentation indexes are shown in Table 1.

[0080] Comparative Example 5 Same as Example 1, the only difference is that according to the traditional fermentation method, 0.9m 3 / h constant speed flow of glucose solution, 0.1m 3 / h uniform flow of nutrient solution, the nutrient solution does not contain a fermentation enhancer, and the fermentation liquid does not enter the fermentation auxiliary device during the fermentation, and the fermentation ends when the content of L-isoleucine in the fermentation liquid is ≥4.3%.

[0081] The fermentation indexes are shown in Table 1.

[0082] Table 1 Fermentation index data in Examples 1 to 2 and Comparative Examples 1 to 5

[0083] The fermentation cycle in Table 1 is calculated from the time when the seed liquid is connected to the fermentation tank. After the fermentation liquid is transferred to the auxiliary device for several cycles, the fermentation ends when the L-isoleucine content in the fermentation liquid reaches more than 4.3%. This fermentation process is regarded as one fermentation cycle.

[0084] The acid production in Table 1 refers to the acid content of L-isoleucine in the fermentation broth obtained from the last fermentation. The volume of the discharged material is the sum of the amount of L-isoleucine separated by the auxiliary device. The conversion rate refers to the amount of acid produced / the amount of glucose 100%, glucose volume is the total amount of glucose used in the fermentation process. Fermentation production intensity refers to the acid production / fermentation cycle / total volume of the fermentation tank equipment; among them, the discharge volume refers to the volume of all materials in the fermentation tank after the fermentation is completed. The total volume of the fermentation tank equipment in the present invention is 75m 3 The total volume of the embodiment and the comparative example of the present invention is 75m 3 Fermentation tank.

[0085] As can be seen from Table 1, in Example 1 and Example 2, the change in fermentation pH reflects the growth of the bacteria. The glucose flow acceleration is adjusted by feedback of the pH change, and the nutrient solution containing the fermentation enhancer is added, so that the fermentation process can be accurately controlled; when the fermentation reaches a certain degree, L-isoleucine is separated in time by the fermentation auxiliary device to avoid feedback inhibition caused by product accumulation, and to avoid L-isoleucine crystallization blocking the pipeline, forcing the fermentation to stop. The separated nutrients and bacteria are returned to the fermentation tank for continued fermentation and cultivation, which improves the fermentation yield and equipment utilization, and improves the fermentation production intensity.

[0086] In comparative example 1, the glucose solution is added at a uniform rate. During the control process, the sugar concentration may be too high or too low. Too high a sugar concentration will inhibit the growth of the bacteria, while too low a sugar concentration will affect the metabolism of the bacteria, further affecting the production and accumulation of L-isoleucine.

[0087] In comparative example 2, the nutrient solution is added at a uniform rate. During the control process, there may be an excess or deficiency of nutrients. If the rate is too high, the bacteria will multiply in large numbers and the conversion rate will be low. If the rate is too low, the growth of the bacteria will be inhibited, the bacteria will decline faster, and the acid production will be lower.

[0088] In comparative example 3, no fermentation enhancer was added, which would result in low metabolic enzyme activity, slow metabolism, prolonged fermentation cycle and low conversion rate.

[0089] Although the fermentation process was precisely controlled in Comparative Example 4, no fermentation auxiliary device was used. When the fermentation cycle was 31 h, a large amount of crystals appeared in the fermenter, blocking the pipeline and affecting the fermentation, and the fermenter was forced to stop halfway.

[0090] Comparative Example 5 follows the traditional fermentation method, which cannot accurately control the fermentation process and does not use fermentation auxiliary equipment. The final output and conversion rate are low, the equipment utilization rate is low, and the production cost is increased.

[0091] Comparative Example 6 The same as Example 1, the only difference is that hexadecyl sulfobetaine is not added to the fermentation enhancer. The fermentation indexes are shown in Table 2.

[0092] Comparative Example 7 Same as Example 1, the only difference is that octadecyl dimethyl betaine is not added to the fermentation enhancer. The fermentation indexes are shown in Table 2.

[0093] Comparative Example 8 Same as Example 1, the only difference is that CGF is not added to the fermentation enhancer. The fermentation indexes are shown in Table 2.

[0094] Comparative Example 9 Same as Example 1, the only difference is that cobalt chloride is not added to the fermentation enhancer. The fermentation indexes are shown in Table 2.

[0095] Comparative Example 10 Same as Example 1, the only difference is that copper chloride is not added to the fermentation enhancer. The fermentation indexes are shown in Table 2.

[0096] Table 2 Fermentation index data in comparative examples 6 to 10

[0097] The fermentation index data of Comparative Examples 6 to 10 are shown in Table 2, and it can be seen that: In comparative examples 6 to 10, when a certain component is missing in the fermentation enhancer, the fermentation period is extended by 3 to 4 hours compared with the example, and the fermentation production intensity and conversion rate are both reduced. This indicates that the fermentation enhancer is the best fermentation enhancer suitable for the fermentation production of L-isoleucine, and the lack of any one component has a significant impact on the fermentation production.

[0098] It can be seen that the technical solution of the present invention has the following advantages: (1) Under the combined action of the fermentation enhancers hexadecyl sulfobetaine and octadecyl dimethyl betaine surfactants, cobalt chloride and copper chloride metal ions and CGF nutrients, the cell membrane permeability of the L-isoleucine fermentation bacteria is better, nutrients quickly enter the cells, the metabolic activity of the bacteria is greatly enhanced, and the L-isoleucine fermentation production efficiency is improved; (2) The pH adjustment rate directly reflects the growth rate of the microorganism. The glucose flow acceleration is adjusted by feedback of the pH change, and the nutrient solution containing the fermentation enhancer is added to accurately control the L-isoleucine fermentation process, so that the bacteria quickly accumulate and produce L-isoleucine, shorten the fermentation cycle, and improve the fermentation conversion rate; (3) By using the fermentation production auxiliary device, the L-isoleucine in the fermentation broth is quickly crystallized and separated from the fermentation broth, and the nutrients and bacteria are re-entered into the fermentation tank for continued fermentation and cultivation, avoiding the feedback inhibition caused by the accumulation of products and the forced cessation of fermentation due to the blockage of the pipeline by L-isoleucine crystals, thereby achieving the goal of increasing the fermentation yield, improving the utilization rate of the fermentation tank and improving the production efficiency.

[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 enhancer for improving the fermentation production efficiency of L-isoleucine, characterized in that: The fermentation enhancer uses water as a solvent and includes components with the following concentrations: 18-20 g / L hexadecyl sulfobetaine, 20-22 g / L octadecyl dimethyl betaine, 0.3-0.5 g / L chlorella growth factor, 2-4 g / L cobalt chloride and 1-2 g / L copper chloride.

2. A method for improving the fermentation production efficiency of L-isoleucine, characterized in that: The steps include: Inoculating Corynebacterium glutamicum into a fermentation medium for fermentation culture, and starting to feed a glucose solution and a nutrient solution when the glucose concentration in the fermentation broth is less than 0.5% (w / v), wherein the nutrient solution contains the fermentation enhancer according to claim 1; during the fermentation culture, liquid ammonia is introduced to maintain the pH value of the fermentation culture at 7.3-7.5, and the volume of the introduced liquid ammonia is determined according to the change in the pH value; When the concentration of L-isoleucine in the fermentation broth reaches 4.3% to 4.6%, the L-isoleucine in the fermentation broth is separated, and the separated fermentation broth is continued to be fermented and cultured.

3. The method for improving the fermentation production efficiency of L-isoleucine according to claim 2, characterized in that: The volume of the glucose solution added is determined according to the volume of liquid ammonia introduced per unit time, and the volume ratio of the liquid ammonia introduced to the glucose solution added per unit time is 1:(15~16); the volume of the nutrient solution added is determined according to the volume of the glucose solution added per unit time, and the volume ratio of the glucose solution added per unit time to the nutrient solution added per unit time is (10~11):

1.

4. The method for improving the fermentation efficiency of L-isoleucine according to claim 2 or 3, characterized in that: The nutrient solution uses water as a solvent and includes the following components in concentrations: 8-10 g / L of the fermentation enhancer according to claim 1, 0.6-0.8 g / L of betaine, 2-4 g / L of magnesium sulfate, 1-2 g / L of potassium dihydrogen phosphate, 13-15 mg / L of biotin, 15-18 mg / L of nicotinamide, 10-12 mg / L of calcium pantothenate, and 20-22 mg / L of thiamine.

5. The method for improving the fermentation efficiency of L-isoleucine according to claim 2, characterized in that: The fermentation temperature is 33-35°C, the fermentation pressure is 0.03-0.05 MPa, and the ventilation rate is 0.3-0.5 m3 / min per cubic meter of fermentation liquid. 3 Air.

6. The method for improving the fermentation efficiency of L-isoleucine according to claim 2, characterized in that: The fermentation medium uses water as a solvent and includes components in the following concentrations: 5-8 g / L yeast powder, 10-15 g / L glucose, 0.2-0.5 g / L magnesium sulfate, 5-8 g / L corn steep liquor, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L ammonium citrate, 10-12 mg / L biotin, 5-7 mg / L nicotinamide and 8-10 mg / L calcium pantothenate.

7. The method for improving the fermentation efficiency of L-isoleucine according to claim 2, characterized in that: The inoculation method of the Corynebacterium glutamicum includes seed liquid; the inoculation amount of the seed liquid is 20% to 25% of the volume of the fermentation medium.

8. The method for improving the fermentation production efficiency of L-isoleucine according to claim 2 or 3, characterized in that: The concentration of the glucose solution is 50% to 55%.

9. The method for improving the fermentation production efficiency of L-isoleucine according to claim 2, characterized in that: During the fermentation culture, L-isoleucine is separated 2 to 3 times in the fermentation broth.

10. The method for improving the fermentation production efficiency of L-isoleucine according to claim 2, characterized in that: The separation method includes passing the fermentation fluid into a separation device.

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