A fermentation intensifier and method for improving the fermentation production efficiency of L-isoleucine

Abstract

The present invention belongs to the technical field of preparing amino acids by microbial fermentation, and specifically relates to a fermentation intensifier and method for improving the fermentation production efficiency of L-isoleucine. The present invention provides a fermentation intensifier for improving the fermentation production efficiency of L-isoleucine, which can promote the rapid growth and reproduction of bacteria and the metabolism of bacteria, and improve the yield of L-isoleucine and the fermentation production efficiency. The present invention also provides a method for improving the fermentation production efficiency of L-isoleucine by using the fermentation intensifier described in the above technical solution. By feeding a glucose solution and a nutrient solution containing the fermentation intensifier, the present invention precisely controls the L-isoleucine fermentation process, enables the bacteria to rapidly accumulate and produce L-isoleucine, shortens the fermentation cycle, and improves the fermentation conversion rate. At the same time, during the fermentation process, L-isoleucine in the fermentation broth is rapidly crystallized and separated from the fermentation broth to avoid the feedback inhibition caused by the accumulation of products, and improve the fermentation production efficiency of L-isoleucine.

Description

Technical Field

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

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

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

[0004] In the prior art, the yield of L-isoleucine prepared by the microbial fermentation method is low, and the utilization rate of fermentation production equipment is low, resulting in low production efficiency. 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 purpose of the present invention is to provide a fermentation intensifier and method for improving the fermentation production efficiency of L-isoleucine. By adding the fermentation intensifier in a fed-batch manner, precisely controlling the fermentation process, improving the metabolic activity of the bacterial cells, and timely separating L-isoleucine in the fermentation broth from the fermentation broth to avoid the feedback inhibition caused by the accumulation of products, the yield of L-isoleucine and the fermentation production efficiency are improved.

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

[0007] The present invention provides a fermentation intensifier for improving the fermentation production efficiency of L-isoleucine. The fermentation intensifier uses water as a solvent and includes components with the following concentrations: 18-20 g / L of cetyl 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.

[0008] The present invention provides a method for improving the fermentation production efficiency of L-isoleucine, including the following steps:

[0009] Corynebacterium glutamicum is inoculated into a fermentation medium for fermentation culture. When the glucose concentration in the fermentation broth < 0.5% (w / v), a glucose solution and a nutrient solution are added dropwise. The nutrient solution contains the fermentation intensifier described in the above technical solution; during the fermentation culture process, 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 of the pH value.

[0010] When the L-isoleucine concentration in the fermentation broth reaches 4.3% - 4.6%, the L-isoleucine in the fermentation broth is separated, and the separated fermentation broth continues for fermentation culture.

[0011] Preferably, the volume of the added glucose solution per unit time is determined according to the volume of the introduced liquid ammonia per unit time, and the volume ratio of the introduced liquid ammonia to the added glucose solution is 1:(15 - 16); the volume of the added nutrient solution per unit time is determined according to the volume of the added glucose solution per unit time, and the volume ratio of the added glucose solution to the added nutrient solution is (10 - 11):1.

[0012] Preferably, the nutrient solution uses water as a solvent and includes components with the following concentrations: 8 - 10 g / L of the fermentation intensifier 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.

[0013] Preferably, the temperature of the fermentation culture is 33 - 35 °C, the pressure of the fermentation culture is 0.03 - 0.05 MPa, and the ventilation volume of the fermentation culture is 0.3 - 0.5 m 3 air per cubic meter of fermentation broth per minute.

[0014] Preferably, the fermentation medium uses water as a solvent and includes components with the following concentrations: 5 - 8 g / L of yeast powder, 10 - 15 g / L of glucose, 0.2 - 0.5 g / L of magnesium sulfate, 5 - 8 g / L of corn steep liquor, 1 - 2 g / L of potassium dihydrogen phosphate, 2 - 4 g / L of ammonium citrate, 10 - 12 mg / L of biotin, 5 - 7 mg / L of nicotinamide, and 8 - 10 mg / L of calcium pantothenate.

[0015] Preferably, the inoculation method of Corynebacterium glutamicum includes a seed solution; the inoculation amount of the seed solution is 20% - 25% of the volume of the fermentation medium.

[0016] Preferably, the concentration of the glucose solution is 50% - 55%.

[0017] Preferably, the number of times of separating L-isoleucine in the fermentation broth during the fermentation culture process is 2 - 3 times.

[0018] Preferably, the separation method includes pressing the fermentation broth into a separation device.

[0019] Advantages of the present invention:

[0020] 1. The present invention provides a fermentation intensifier for improving the fermentation production efficiency of L-isoleucine. The cetylsulfobetaine and octadecyl dimethyl betaine in the fermentation intensifier are quaternary ammonium salt surfactants, which can change the permeability of the cell membrane of the bacteria, promote the transport of various nutrients into the bacterial cells, and at the same time promote the release of L-isoleucine to the outside of the cells; Chlorella growth factor (CGF) contains rich nucleoproteins, nucleic acids, ribonucleic acids, deoxyribonucleic acids, various vitamins, amino acids, polysaccharide components, complex protein bodies, enzymes, glycoproteins, etc., which can improve the cell metabolic activity and accelerate the conversion of glucose to synthesize L-isoleucine; The metal ions cobalt chloride and copper chloride can not only assist CGF to be transported into the bacterial cells through transport proteins, but also improve the metabolic enzyme activity of the bacteria and promote the growth and reproduction of the bacteria; Under the combined action of the cetylsulfobetaine 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, and the metabolic activity of the bacteria is greatly enhanced, improving the fermentation production efficiency of L-isoleucine.

[0021] 2. The present invention also provides a method for improving the fermentation production efficiency of L-isoleucine. The present invention adjusts the feeding amount of liquid ammonia through the change of pH, and further feedback regulates the feeding of glucose solution and the feeding of nutrient solution containing the fermentation intensifier described in the above technical solution, accurately controlling the content of nutrient components and the amount of synthesis raw materials of L-isoleucine in the L-isoleucine fermentation process, enabling the bacteria to quickly accumulate and produce L-isoleucine, shortening the fermentation cycle and improving the fermentation conversion rate.

[0022] 3. The present invention quickly and cryogenically crystallizes and separates L-isoleucine from the fermentation broth through a separation device, and then uses nutrients and bacteria to continue fermentation culture, avoiding the feedback inhibition caused by the accumulation of products and the forced stop of fermentation due to the blockage of the pipeline by L-isoleucine crystals, thereby achieving the effect of improving the fermentation yield and conversion rate, and ultimately improving the fermentation production efficiency of L-isoleucine. Description of the Drawings

[0023] Figure 1 It is a schematic structural diagram of an auxiliary device for improving the amino acid production efficiency applied in the present invention;

[0024] Figure 2 is Figure 1 an enlarged view of M in

[0025] Figure 3 Schematic diagram of the installation position of the blocking component;

[0026] Figure 4 Schematic diagram of the structure of the coil pipe;

[0027] Figure 5 Schematic diagram of the structure of the support component;

[0028] Figure 6 Schematic diagram of the structure of the spraying component;

[0029] Figures 1 to 6 In:

[0030] 1 - tank body, 2 - three - way pipe, 21 - feed valve, 22 - cleaning valve, 23 - blocking component, 231 - pipe cover, 232 - movable baffle, 233 - fixed baffle, 234 - handle, 24 - feed pipeline, 25 - cleaning pipeline, 26 - tank body connecting pipe, 27 - rubber layer, 3 - coil pipe, 31 - coil pipe inlet connecting pipe, 32 - coil pipe outlet connecting pipe, 33 - coil pipe connecting pipe, 4 - support component, 41 - fixing frame, 42 - sliding frame, 43 - support frame, 44 - fixing plate, 5 - spraying component, 6 - tank body discharge pipe, 7 - fermentation broth discharge pipe valve, 8 - L - isoleucine discharge pipe valve, 51 - spraying port;

[0031] Figure 7 Fermentation flow chart for improving the fermentation production efficiency of L - isoleucine. Specific implementation mode

[0032] The present invention provides a fermentation intensifier for improving the fermentation production efficiency of L - isoleucine, comprising the following concentration components:

[0033] Cetyl sulfobetaine 18 - 20 g / L, octadecyl dimethyl betaine 20 - 22 g / L, CGF 0.3 - 0.5 g / L, cobalt chloride 2 - 4 g / L, and copper chloride 1 - 2 g / L.

[0034] As an optional implementation mode, the fermentation intensifier provided by the present invention comprises cetyl sulfobetaine 18 - 20 g / L, and it can also be 18.5 - 19.5 g / L. As an optional implementation mode, the fermentation intensifier provided by the present invention comprises octadecyl dimethyl betaine 20 - 22 g / L, and it can also be 20.5 - 21.5 g / L. The cetyl sulfobetaine and octadecyl dimethyl betaine of the present invention can change the permeability of the cell membrane of the bacteria, promote the transport of various nutrients such as CGF into the bacterial cells, and at the same time promote the release of L - isoleucine to the outside of the cells.

[0035] As an alternative embodiment, the fermentation enhancer provided by the present invention may include 0.3 - 0.5 g / L of CGF, or may be 0.35 - 0.45 g / L. CGF provides rich nutrients for the bacterial cells, improves the cell metabolic activity, and accelerates the conversion of glucose to synthesize L-isoleucine.

[0036] As an alternative embodiment, the fermentation enhancer provided by the present invention includes 2 - 4 g / L of cobalt chloride, or may be 2.5 - 3.5 g / L. The fermentation enhancer provided by the present invention includes 1 - 2 g / L of copper chloride, or may be 1.2 - 1.6 g / L. The cobalt chloride and copper chloride of the present invention can not only assist CGF to be transported into the bacterial cells through the transporter protein, but also improve the metabolic enzyme activity of the bacteria and promote the growth and reproduction of the bacteria.

[0037] Under the combined action of cetyl sulfobetaine, octadecyl dimethyl betaine, CGF, cobalt chloride and copper chloride in the fermentation enhancer provided by the present invention, the cell membrane permeability of the L-isoleucine fermentation bacteria is better, nutrients quickly enter the cells, and the metabolic activity of the bacteria is greatly enhanced, improving the fermentation production efficiency of L-isoleucine.

[0038] As an alternative embodiment, the fermentation enhancer of the present invention uses water as a solvent.

[0039] The present invention has no special limitation on the sources of the components in the fermentation enhancer, and those well-known to those skilled in the art can be used.

[0040] The present invention provides a method for improving the fermentation production efficiency of L-isoleucine, including the following steps:

[0041] Inoculate Corynebacterium glutamicum into the fermentation medium for fermentation culture. When the glucose concentration in the fermentation broth < 0.5% (w / v), start to feed a glucose solution and a nutrient solution. The nutrient solution contains the fermentation enhancer described in the above technical solution; during the fermentation culture process, introduce liquid ammonia to maintain the pH value of the fermentation culture at 7.3 - 7.5, and determine the volume of the introduced liquid ammonia according to the change of the pH value;

[0042] When the L-isoleucine concentration in the fermentation broth reaches 4.3% - 4.6%, separate the L-isoleucine in the fermentation broth, and continue the fermentation culture of the separated fermentation broth.

[0043] In the present invention, there is no special limitation on the strain type of Corynebacterium glutamicum, and any strain type of Corynebacterium glutamicum commonly used in the art for fermenting and preparing L-isoleucine can be used.

[0044] As an alternative embodiment, the seed liquid of Corynebacterium glutamicum is inoculated into a fermentation medium for fermentation culture. The present invention does not particularly limit the preparation method of the seed liquid, and conventional methods can be used. The present invention does not particularly limit the inoculation method, and conventional methods can be used. The inoculation amount of the seed liquid in the present invention can be 20% - 25% of the volume of the fermentation medium, or can also be 24% - 25%. As an embodiment, the fermentation medium in the present invention can use water as a solvent and includes components with 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; in the examples of the present invention, the fermentation medium can include yeast powder 6 g / L, glucose 12 g / L, magnesium sulfate 0.3 g / L, corn steep liquor 5 g / L, potassium dihydrogen phosphate 1 g / L, ammonium citrate 3 g / L, biotin 11 mg / L, nicotinamide 5 mg / L, and calcium pantothenate 8 mg / L; or can also include yeast powder 8 g / L, glucose 15 g / L, magnesium sulfate 0.2 g / L, corn steep liquor 7 g / L, potassium dihydrogen phosphate 1.5 g / L, ammonium citrate 4 g / L, biotin 12 mg / L, nicotinamide 7 mg / L, and calcium pantothenate 10 mg / L.

[0045] Generally, the optimal growth pH of microorganisms is neutral. However, since acidic substances will be produced during their metabolic process, an acidic pH of the fermentation broth will affect the growth and reproduction of microorganisms. Therefore, it is usually necessary to introduce liquid ammonia during the microbial fermentation process to maintain the pH value at neutral. During the fermentation culture process of the present invention, liquid ammonia is introduced to adjust the pH value of the fermentation broth to 7.3 - 7.5, or can also be 7.4. As an alternative embodiment, the temperature of the fermentation culture in the present invention is 33 - 35 °C, or can also be 34 - 35 °C. The pressure of the fermentation culture in the present invention is 0.03 - 0.05 MPa, or can also be 0.05 MPa. As an alternative embodiment, the fermentation culture in the present invention is carried out under ventilation conditions, and the ventilation rate can be 0.3 - 0.5 m 3 air / m 3 fermentation broth / min, or can also be 0.5 m 3 air / m 3 fermentation broth / min.

[0046] During the fermentation culture process of the present invention, the pH value is maintained at 7.3 - 7.5, and the pH value of the fermentation culture is adjusted to 7.3 - 7.5 by introducing liquid ammonia. Maintaining the pH value condition of the fermentation culture in the present invention helps Corynebacterium glutamicum to grow and reproduce to produce L-isoleucine. It is preferred to introduce liquid ammonia from the beginning, and the liquid ammonia rate is determined according to the change of pH to ensure that the system pH value is maintained within the range of 7.3 - 7.5.

[0047] After the start of fermentation culture, the present invention preferably monitors the mass concentration of glucose in the fermentation broth. When the mass concentration of glucose in the fermentation broth of the present invention is first <0.5% (w / v), glucose solution and nutrient solution are fed. The present invention preferably continues to add them after starting to feed glucose and nutrient solution. In the present invention, the nutrient solution contains the fermentation intensifier described in the above technical solution. Glucose is the substrate for Corynebacterium glutamicum to ferment and produce L-isoleucine, and the nutrient solution provides nutrients for the growth and reproduction of Corynebacterium glutamicum.

[0048] The feeding rate of the glucose solution in the present invention is feedback-regulated according to the change of pH, that is, determined according to the rate of introduced liquid ammonia. The feeding rate of the nutrient solution is determined according to the feeding rate of the 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 feeding rate of liquid ammonia is controlled by a flow meter according to the change of the pH value of the fermentation broth. The feeding rates of the glucose solution and the liquid ammonia solution in the present invention are also interlocked and controlled by a flow meter. The present invention determines the volume of the glucose solution fed per unit time according to the volume of the liquid ammonia introduced per unit time. The ratio of the flow rate of the liquid ammonia to the flow rate of the glucose is 1:15 - 16; that is, the volume ratio of the liquid ammonia to the glucose solution fed per unit time in the present invention 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 the glucose is 15 - 16m 3 / h. In the present invention, the nutrient solution is fed while the glucose solution is fed, and the feeding rates of the glucose solution and the nutrient solution in the present invention are also interlocked and controlled by a flow meter. For example, when the instantaneous flow rate of the glucose is 10 - 11m 3 / h, the instantaneous flow rate of the nutrient solution is 1m 3 / h. As an alternative embodiment, the present invention determines the volume of the nutrient solution fed per unit time according to the volume of the glucose solution fed per unit time. The volume ratio of the glucose solution to the nutrient solution per unit time is (10 - 11):1, and it can also be 10.5:1. As an alternative embodiment, the concentration of the fed glucose solution in the present invention is 50% - 55% (w / v), and it can also be 53% - 55% (w / v).

[0049] During the fermentation process of the present invention, the change in pH, i.e., the feeding frequency of liquid ammonia, directly reflects the growth rate of microorganisms. At the beginning of fermentation, the growth of microorganisms is slow, the metabolic rate is low, and the amount of acidic substances produced during metabolism is also small. The regulation rate of pH is slow, that is, the feeding frequency of liquid ammonia is relatively low. As the fermentation cycle extends, the microorganisms grow vigorously, the metabolic rate increases, and the amount of acidic substances produced during metabolism also increases accordingly. The regulation rate of pH accelerates, that is, the feeding frequency of liquid ammonia increases. By feedback regulating the acceleration of sugar flow through the change in pH, the fermentation process of L-isoleucine is precisely controlled. At the same time, a fermentation intensifier is fed to provide nutrients for the reproduction and metabolism of microorganisms, making the metabolism of the bacterial cells more conducive to the accumulation and production of L-isoleucine, shortening the fermentation cycle, and improving the fermentation conversion rate.

[0050] In the present invention, when the glucose concentration in the fermentation broth < 0.5% (w / v), a glucose solution and a nutrient solution are fed. The nutrient solution contains the fermentation intensifier described in the above technical solution.

[0051] As an alternative embodiment, the nutrient solution uses water as a solvent and includes components with the following concentrations: the fermentation intensifier described in the above technical solution 8 - 10 g / L, betaine 0.6 - 0.8 g / L, magnesium sulfate 2 - 4 g / L, potassium dihydrogen phosphate 1 - 2 g / L, biotin 13 - 15 mg / L, nicotinamide 15 - 18 mg / L, calcium pantothenate 10 - 12 mg / L, and thiamine 20 - 22 mg / L.

[0052] As an alternative 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 - 0.8 g / L, and can also be 0.65 - 0.75 g / L. The components of the nutrient solution provided by the present invention include magnesium sulfate 2 - 4 g / L, and can also be 2.5 - 3.5 g / L. The components of the nutrient solution provided by the present invention include potassium dihydrogen phosphate 1 - 2 g / L, and can also be 1.3 - 1.8 g / L. The components of the nutrient solution provided by the present invention include biotin 13 - 15 mg / L, and can also be 13.5 - 14.5 mg / L. The components of the nutrient solution provided by the present invention include nicotinamide 15 - 18 mg / L, and can also be 15.5 - 17.5 mg / L. The components of the nutrient solution provided by the present invention include calcium pantothenate 10 - 12 mg / L, and can also be 10.5 - 11.5 mg / L. The components of the nutrient solution provided by the present invention include thiamine 20 - 22 mg / L, and can also be 20.5 - 21.5 mg / L.

[0053] Generally, the solubility of L-isoleucine is relatively low. As the fermentation culture progresses, the concentration of L-isoleucine in the fermentation broth gradually increases. When the content in the fermentation broth ≥ 4.5%, crystallization begins. If the crystallization is severe, it is easy to block the fermenter and affect fermentation production. Moreover, the accumulation of products during the fermentation process often causes feedback repression. Usually, when the fermentation reaches 4.0% - 4.5% of L-isoleucine content, the fermentation ends. At this time, stopping the tank not only results in a low yield, but also a low utilization rate of the fermentation production equipment and low production efficiency. Therefore, in the present invention, 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 separated fermentation broth continues to be fermented and cultured. The separation method described in the present invention may include pumping the fermentation broth into a separation device. The separation device can enable the L-isoleucine in the fermentation broth to rapidly crystallize at a low temperature, quickly separate the fermentation broth, and the bacteria and nutrients in the separated fermentation broth are returned to the fermentation equipment to continue fermentation and culture, achieving the purpose of increasing 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 the tank body. The auxiliary device includes a plurality of coiled pipes 3 arranged at equal intervals from top to bottom in the tank body. Adjacent coiled pipes 3 are connected to each other, and adjacent two layers of coiled pipes 3 are arranged in a staggered manner; a spraying component is arranged in the tank body, and the spraying component is located above the coiled pipes; the spraying component has a cross structure, and a plurality of spraying ports are arranged at the bottom of the spraying component.

[0054] As an alternative embodiment, refer to Figure 1 and Figure 3 , the auxiliary device for improving the production efficiency of amino acids provided by the present invention is arranged in the tank body 1. The auxiliary device includes a plurality of coiled pipes 3 arranged at equal intervals from top to bottom in the tank body 1. Adjacent coiled pipes 3 are connected to each other, and adjacent two layers of coiled pipes 3 are arranged in a staggered manner. The plurality of coiled pipes 3 are connected by a coiled pipe connecting pipe 33; the coiled pipe 3 at the bottommost is connected with a coiled pipe inlet connecting pipe 31, and one end of the coiled pipe inlet connecting pipe 31 passes through the side wall of the tank body 1; the coiled pipe 3 at the topmost is connected with a coiled pipe outlet connecting pipe 32, and one end of the coiled pipe outlet connecting pipe 32 passes through the side wall of the tank body 1. Preferably, the coiled pipe inlet connecting pipe 31 is located on the opposite side of the coiled pipe outlet connecting pipe 32.

[0055] As an alternative embodiment, the coiled pipe 3 is fixed in the tank body 1 through a support member 4. The support member 4 is internally provided with a hollow structure, and the coiled pipe connecting pipe 33 between adjacent two layers of coiled pipes 3 can be connected through the hollow structure of the support member 4. Such a setting not only reduces the weight of the support member 4, but also makes the connection between adjacent two layers of coiled pipes 3 smoother and more convenient. In order to adapt to the staggered arrangement structure of adjacent two layers of coiled pipes 3, the support member 4 is set as a telescopic structure. The structure is shown in Figure 5, Specifically, the support member 4 includes a fixed frame 41. A sliding frame 42 is sleeved inside the fixed frame 41. The end of the sliding frame 42 is connected to a support frame 43. The sliding distance of the sliding frame 42 can be adjusted according to the position requirement of the coil pipe 3, so that the support frame 43 can more stably support the coil pipe 3. An fixing plate 44 is further arranged on the support member 4. The fixing plate 44 is of an arc structure, and its radian is adapted to the radian of the side wall of the tank body 1. The fixing plate 44 can be connected to the tank body 1 by bolts or can be pasted on the tank body 1 by using existing pasting techniques.

[0056] The coil pipe 3 can be fixed on the inner wall of the tank body 1 through the support member 4 to prevent the coil pipe 3 from shaking and causing danger during operation.

[0057] As an optional implementation manner, a detachable blocking member 23 is arranged at the central position of the tee pipe 2. Refer to Figures 1 to 3 , the blocking member 23 includes a pipe cover 231. A handle 234 is arranged at the top of the pipe cover 231. The pipe cover 231 is connected to a fixed blocking plate 233. The height of the fixed blocking plate 233 is 1 / 2 of the pipe diameter of the tee pipe 2.

[0058] As an optional implementation manner, refer to Figure 1 and Figure 6 , a spraying member 5 is arranged inside the tank body 1 provided by the present invention. The spraying member 5 is located above the coil pipe 3. The spraying member 5 is of a cross structure. A plurality of spraying ports 51 are arranged at the bottom of the spraying member 5. By arranging the spraying member in a cross structure, the fermentation broth containing amino acids can be sprayed more evenly in the entire tank body 1.

[0059] As an optional implementation manner, the top of the spraying member 5 is communicated with the third pipe orifice of the tee pipe 2. Refer to Figures 1 to 3, the other two pipe orifices of the tee 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 and separable barrier member 23 is arranged at the central position of the tee pipe 2. The barrier member 23 includes a pipe cover 231. A handle 234 is arranged at the top of the pipe cover 231. The pipe cover 231 is connected with a fixed barrier plate 233. A movable barrier plate 232 is hinged to the bottom of the fixed barrier plate 233. The movable barrier plate 232 extends into the third pipe orifice of the tee pipe 2. The height of the fixed barrier plate 233 is 1 / 2 of the pipe diameter of the tee pipe 2. A corrosion-resistant rubber layer 27 is arranged at the third pipe orifice of the tee pipe 2. In order to prevent the movable barrier plate 232 from swinging left and right and causing collision damage to the tee pipe 2, the corrosion-resistant rubber layer 27 is arranged at the third pipe orifice of the tee pipe 2. The pipe orifice of the tee pipe 2 where the feed valve 21 is arranged is connected with a feed pipeline 24. The pipe orifice of the tee pipe 2 where the cleaning valve 22 is arranged is connected with a cleaning pipeline 25. The third pipe orifice of the tee pipe 2 is connected with a tank body connecting pipe 26. The bottom of the tank body 1 is connected with a tank body discharge pipe 6. A fermentation liquid discharge pipe and an amino acid discharge pipe are connected to the tank body discharge pipe 6. A fermentation liquid discharge pipe valve 7 is arranged on the fermentation liquid discharge pipe. An L-isoleucine discharge pipe valve 8 is arranged on the amino acid discharge pipe. A sight glass is arranged on the tank body 1. Through the fermentation production auxiliary device of the present invention, L-isoleucine in the fermentation liquid is quickly crystallized and separated from the fermentation liquid, so that the concentration of L-isoleucine in the fermentation liquid is reduced, and nutrients, bacterial cells, etc. re-enter the fermentation tank for continuous fermentation culture, avoiding the feedback inhibition caused by the accumulation of products and the forced stop of fermentation due to the blockage of the pipeline by L-isoleucine crystals, thereby achieving the purpose of increasing the fermentation yield, improving the utilization rate of the fermentation tank and the production efficiency.

[0060] As an alternative embodiment, Corynebacterium glutamicum is inoculated into a fermentation medium for fermentation culture. When the concentration of L-isoleucine in the obtained fermentation broth reaches 4.3% - 4.6% for the first time, L-isoleucine in the fermentation broth is separated for the first time. The separated fermentation broth is returned to the fermentation equipment for continuous fermentation culture, and the fermentation culture is still carried out at the temperature, ventilation rate, and pressure described in the above technical solution. At the same time, a glucose solution, a nutrient solution, and liquid ammonia are added dropwise during the fermentation culture process according to the above technical solution. When the concentration of L-isoleucine in the fermentation broth reaches 4.3% - 4.6% for the second time, L-isoleucine in the fermentation broth is separated for the second time. The separated fermentation broth is returned to the fermentation equipment for continuous fermentation culture. When the concentration of L-isoleucine in the fermentation broth reaches 4.3% - 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, only a glucose solution and a nutrient solution are added dropwise, and the components in the nutrient solution can provide nutrients for the growth of the bacterial cells and thus synthesize L-isoleucine. The number of times of separating L-isoleucine in the fermentation broth during the fermentation culture process of the present invention can be 2 - 3 times. If the separation is continued for the fourth time and above, the vitality of the bacterial cells declines significantly, and the conversion rate and fermentation production intensity are greatly reduced.

[0061] 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 feedback-regulates the flow rate of the glucose solution through the change of pH, and adds dropwise a nutrient solution containing a fermentation intensifier to precisely control the L-isoleucine fermentation process, enabling the bacterial cells to rapidly accumulate and produce L-isoleucine, shortening the fermentation cycle, and improving the fermentation conversion rate. At the same time, by using a fermentation production auxiliary device, L-isoleucine in the fermentation broth is rapidly crystallized and separated from the fermentation broth, and the nutrients and bacterial cells, etc. re-enter the fermentation tank for continuous fermentation culture, thereby achieving an increase in fermentation yield, an improvement in the utilization rate of the fermentation tank, and production efficiency.

[0062] To further illustrate the present invention, the technical solution provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0063] The present invention has no special limitation on the strain type of Corynebacterium glutamicum used in the following examples and comparative examples, and any strain type conventionally used in the field for fermenting and preparing L-isoleucine can be adopted. The present invention has no special limitation on 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 .

[0064] The detection method for the L-isoleucine content involved in the following examples and comparative examples is paper chromatography, and the method is as follows:

[0065] Dilute the fermentation broth first and then conduct the measurement. 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 filter paper of 30*25 cm. The spotting line is 1.5 cm away from the bottom end of the filter paper, with a spacing of 2.0 cm, and the spotting volume is 1 μL. At the same time, spot a standard sample with an amino acid concentration of 2.0%.

[0066] Place the spotted filter paper in a chromatography tank pre-added with a developing agent, and develop it by the ascending method at 25°C. Take it out and dry it when the leading edge of the developing agent reaches 1 cm from the upper end of the filter paper. Spray the developed and dried filter paper with a developer, put it in an oven for heating and coloring, dry it at 105°C for 5 min, and purple-red amino acid spots will appear on the filter paper. Cut off the purple-red spots that appear on the filter paper and soak them in 5 mL of an eluent for 40 min (cover the test tube mouth to prevent ethanol from volatilizing), and measure the absorbance at 506 nm. Use the absorbance value measured with the standard sample to make a standard curve, and then find the L-isoleucine content of the measured dilution on the standard curve. Then, multiply the L-isoleucine content of the dilution by the dilution factor 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 developer is a 0.5% ninhydrin acetone solution by mass concentration. 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.

[0067] The strain used in the following examples and comparative examples of the present invention is the same strain of Corynebacterium glutamicum.

[0068] The Corynebacterium glutamicum seed liquid involved in the following examples and comparative examples is diluted 50 times with water, and the absorbance OD is detected at a wavelength of 600 nm in a spectrophotometer for the dilution 600 When OD 600 reaches 0.6 - 0.8, inoculate the Corynebacterium glutamicum seed liquid into the fermentation medium; the OD 600 of the Corynebacterium glutamicum seed liquid used in different examples and comparative examples is the same.

[0069] The fermentation flow chart for improving the fermentation production efficiency of L-isoleucine is shown in Figure 7 .

[0070] Example 1

[0071] A method for improving the fermentation production efficiency of L-isoleucine, the steps are as follows:

[0072] 1. The Corynebacterium glutamicum seed liquid is inoculated into the fermentation medium at an inoculation amount of 24% by volume for fermentation culture. The fermentation culture consists of the first fermentation, the second fermentation, and the third fermentation, and the first fermentation, the second fermentation, and the third fermentation are carried out in sequence.

[0073] (1) The first fermentation culture

[0074] Parameters: Control the temperature of the fermentation culture at 35°C, and control the pressure of the fermentation tank for the fermentation culture at 0.05 MPa. The fermentation culture is carried out under ventilation conditions, and the ventilation volume is 0.5 m 3 air per cubic meter of fermentation broth per minute (i.e., the ventilation volume is 0.5 m 3 air / m 3 fermentation broth / min). The flow rate of liquid ammonia is measured using a flow meter, and liquid ammonia is introduced to adjust the pH value of the entire fermentation process to 7.4, that is, the flow rate of liquid ammonia is confirmed by feedback according to the change in the pH value of the fermentation broth. At the same time, the glucose concentration in the fermentation broth is monitored in real time. When the glucose concentration in the fermentation broth is first detected to be less than 0.5% (w / v), a glucose solution and a nutrient solution are added continuously throughout the fermentation process. The flow meter flow rate of liquid ammonia and the flow meter flow rate of the glucose solution are interlocked and controlled. For example, if the instantaneous flow rate of liquid ammonia is 0.1 m 3 / h, then the instantaneous flow rate of glucose is 1.5 m 3 / h, that is, the volume ratio of the liquid ammonia solution to the added glucose solution is controlled at 1:15 per unit time. A nutrient solution is added while adding the glucose solution, and the flow meter flow rate of the glucose solution and the flow meter flow rate of the nutrient solution are interlocked and controlled. For example, if the instantaneous flow rate of glucose is 1.5 m 3 / h, then the instantaneous flow rate of the nutrient solution is 0.15 m 3 / h, that is, the volume ratio of the added glucose solution to the nutrient solution is controlled at 10:1 per unit time. The concentration of the added glucose solution is 55% (w / v).

[0075] Separation: When the L-isoleucine content in the fermentation broth obtained from the first fermentation culture is 4.3%, the specific process of pressing the fermentation broth into the fermentation production auxiliary device by differential pressure is as follows: Open the feed valve 21 on the feed pipe 24, and at the same time open the fermentation broth outlet pipe valve 7. At this time, close the cleaning valve 22, and pass the fermentation broth containing L-isoleucine through the feed pipe 24. Under the action of the movable partition plate 232, the fermentation broth containing L-isoleucine is smoothly introduced into the tank body 1, and the movable partition plate 232 can prevent the fermentation broth containing L-isoleucine from entering the cleaning pipe 25.

[0076] The fermentation broth containing L-isoleucine is evenly sprayed onto the coil pipes 3 of each layer through multiple spray nozzles 51 on the spraying component 5. At this time, cold water is introduced into the coil pipes 3, and L-isoleucine quickly condenses and precipitates on the coil pipes 3 when encountering cold, realizing the rapid separation of L-isoleucine from the fermentation broth. The adjacent two layers of coil pipes 3 are arranged staggeredly. Such an arrangement can enable the coil pipes 3 to contact the fermentation broth containing amino acids over a larger area, greatly increasing the amount of amino acids condensed on the coil pipes 3 when encountering cold. When L-isoleucine in the fermentation broth passes through the coil pipes with a lower temperature, it quickly crystallizes and adheres to the coil pipes. The nutrients and bacteria in the remaining fermentation broth after condensation pass through the fermentation broth outlet pipe connected to the tank body discharge pipe 6, and the fermentation broth outlet valve 7 is opened, and then enter the fermentation tank again to continue the second fermentation. An L-isoleucine outlet valve 8 is provided on the L-isoleucine outlet pipe.

[0077] After the feeding is completed, the feeding valve 21 and the fermentation broth outlet valve 7 are closed, hot water or steam is introduced into the coil pipes 3, the temperature of the coil pipes 3 rises, the L-isoleucine attached to the coil pipes 3 dissolves, 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 from the tank body 1 and then enter the subsequent production process.

[0078] At the end of production, caustic solution or acid solution can also be introduced through the cleaning valve 22 to clean the tank body 1 and the coil pipes 3.

[0079] In order to facilitate viewing the situation inside the tank body 1, a sight glass can be provided on the tank body 1, and a manhole can also be provided for the operator to check and clean the crystals remaining in the tank body.

[0080] (2)Secondary fermentation culture: The parameters are the same as those of the first fermentation culture.

[0081] Separation: When the L-isoleucine content in the fermentation broth obtained from the secondary fermentation culture reaches 4.3%, the rapid separation of L-isoleucine is realized again through the fermentation production auxiliary device. The separation parameters are the same as those in the separation in step (1), and the nutrients and bacteria in the fermentation broth continue the third fermentation.

[0082] (3)Tertiary fermentation culture: The parameters are the same as those of the first fermentation culture.

[0083] When the L-isoleucine content in the fermentation broth obtained from the tertiary fermentation reaches 4.3%, the fermentation culture ends. The fermentation indexes are shown in Table 1.

[0084] Among them, the fermentation medium uses water as a solvent, and the component concentrations are as follows: yeast powder 6 g / L, glucose 12 g / L, magnesium sulfate 0.3 g / L, corn steep liquor 5 g / L, potassium dihydrogen phosphate 1 g / L, ammonium citrate 3 g / L, biotin 11 mg / L, nicotinamide 5 mg / L, and calcium pantothenate 8 mg / L.

[0085] The nutrient solution uses water as the solvent, and the component concentrations are as follows: betaine 0.7 g / L, magnesium sulfate 2 g / L, potassium dihydrogen phosphate 1 g / L, biotin 13 mg / L, nicotinamide 16 mg / L, calcium pantothenate 11 mg / L, thiamine 20 mg / L, and fermentation enhancer 8 g / L; the fermentation enhancer uses water as the solvent, and the component concentrations are as follows: cetylsulfobetaine 18 g / L, octadecyldimethylbetaine 20 g / L, CGF 0.3 g / L, cobalt chloride 2 g / L, copper chloride 1 g / L.

[0086] Example 2

[0087] A method for improving the fermentation production efficiency of L-isoleucine, the steps are as follows:

[0088] Inoculate the Corynebacterium glutamicum seed liquid into the fermentation medium at a volume ratio of 25% of the medium volume for fermentation culture. The fermentation culture consists of the first fermentation, the second fermentation, and the third fermentation, and the first fermentation, the second fermentation, and the third fermentation are carried out in sequence.

[0089] (1) The first fermentation culture:

[0090] Parameters: Control the fermentation culture temperature at 34 °C, the pressure at 0.04 MPa, and the ventilation rate at 0.4 m 3 air per cubic meter of fermentation broth per minute (i.e., the ventilation rate is 0.4 m 3 air / m 3 fermentation broth / min). The flow rate of liquid ammonia is measured using a flow meter, and liquid ammonia is introduced to adjust the pH value of the entire fermentation process to 7.3, that is, the flow rate of liquid ammonia is confirmed by feedback according to the change of the pH value of the fermentation broth. At the same time, the glucose concentration in the fermentation broth is monitored in real time. When the glucose concentration in the fermentation broth is first detected to be less than 0.5% (w / v), start to feed glucose solution and nutrient solution continuously throughout the fermentation process. The flow meter flow rate of liquid ammonia and the flow meter flow rate of glucose solution are interlocked. The instantaneous flow rate of liquid ammonia is 1 m 3 / h, and the instantaneous flow rate of glucose is 16 m 3 / h. That is, the volume ratio of liquid ammonia solution to fed glucose solution is controlled at 1:16 per unit time. While feeding the glucose solution, feed the nutrient solution. The flow meter flow rate of the glucose solution and the flow meter flow rate of the nutrient solution are interlocked. The instantaneous flow rate of the nutrient solution is 1 m 3 / h, and the instantaneous flow rate of glucose is 11 m 3 / h. That is, the volume ratio of the fed glucose solution to the nutrient solution is controlled at 11:1 per unit time. The concentration of the fed glucose solution is 53% (w / v).

[0091] 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 by differential pressure. The specific process of pressing the fermentation broth into the fermentation production auxiliary device by differential pressure is the same as the separation in step (1) of Example 1.

[0092] (2) Second fermentation culture:

[0093] Parameters: The same as those in the first fermentation culture.

[0094] Separation: When the L-isoleucine content in the fermentation broth obtained from the second fermentation culture reaches 4.3%, the rapid separation of L-isoleucine is realized again through the fermentation production auxiliary device. The separation parameters are the same as the separation in step (1) of Example 1, and the nutrients and bacteria in the fermentation broth continue the third fermentation.

[0095] (3) Third fermentation culture:

[0096] Parameters: The same as those in the first fermentation culture.

[0097] Separation: When the L-isoleucine content in the fermentation broth obtained from the third fermentation reaches 4.4%, the fermentation ends. The fermentation indexes are shown in Table 1.

[0098] Among them, the fermentation medium uses water as a solvent, and the component concentrations are as follows: yeast powder 8 g / L, glucose 15 g / L, magnesium sulfate 0.2 g / L, corn steep liquor 7 g / L, potassium dihydrogen phosphate 1.5 g / L, ammonium citrate 4 g / L, biotin 12 mg / L, nicotinamide 7 mg / L, and calcium pantothenate 10 mg / L.

[0099] The nutrient solution uses water as a solvent, and the component concentrations are as follows: betaine 0.8 g / L, magnesium sulfate 3 g / L, potassium dihydrogen phosphate 2 g / L, biotin 14 mg / L, nicotinamide 17 mg / L, calcium pantothenate 12 mg / L, thiamine 21 mg / L, and fermentation enhancer 10 g / L; the fermentation enhancer uses water as a solvent, and the component concentrations are as follows: cetylsulfobetaine 20 g / L, octadecyldimethylbetaine 22 g / L, CGF 0.5 g / L, cobalt chloride 4 g / L, and copper chloride 2 g / L.

[0100] Comparative Example 1

[0101] Same as Example 1, the only difference is that the flow rate of the glucose solution added is a uniform flow rate, and the flow rate is selected as 0.9 m 3 / h, and other conditions remain unchanged. The fermentation indexes are shown in Table 1.

[0102] Comparative Example 2

[0103] Same as Example 1, the only difference is that the flow rate of the nutrient solution added is a uniform flow rate, and the flow rate is selected as 0.1 m3 / h, with other conditions remaining unchanged. The fermentation indexes are shown in Table 1.

[0104] Comparative Example 3

[0105] Same as Example 1, the only difference is that the fed nutrient solution does not contain a fermentation enhancer. The fermentation indexes are shown in Table 1.

[0106] Comparative Example 4

[0107] Same as Example 1, the only difference is that during fermentation, the fermentation broth does not enter the fermentation auxiliary device, and fermentation ends when the content of L-isoleucine in the fermentation broth ≥ 4.3%. The fermentation indexes are shown in Table 1.

[0108] Comparative Example 5

[0109] Same as Example 1, the only difference is that in accordance with the traditional fermentation method, glucose solution is fed at a uniform speed of 0.9 m 3 / h, and nutrient solution is fed at a uniform speed of 0.1 m 3 / h. The nutrient solution does not contain a fermentation enhancer, and during fermentation, the fermentation broth does not enter the fermentation auxiliary device. Fermentation ends when the content of L-isoleucine in the fermentation broth ≥ 4.3%.

[0110] The fermentation indexes are shown in Table 1.

[0111] Table 1 Fermentation Index Data in Examples 1-2 and Comparative Examples 1-5

[0112]

[0113] In Table 1, the fermentation cycle is calculated from the time when the seed liquid is introduced into the fermentation tank. After the fermentation broth is transferred to the auxiliary device and circulated several times, and finally when the content of L-isoleucine in the fermentation broth reaches more than 4.3%, fermentation ends, and this fermentation process is regarded as a fermentation cycle.

[0114] The acid production amount in Table 1 refers to the acid content of L-isoleucine in the fermentation broth obtained in the last fermentation. The sum of the discharging volume and the amount of L-isoleucine separated by the auxiliary device. The conversion rate is the acid production amount / glucose amount ×100%, and the glucose amount is the total amount of glucose utilized during the fermentation process. The fermentation production intensity is the acid production amount / fermentation cycle / total volume of the fermentation tank equipment; among them, the discharging volume is the volume of all materials in the fermentation tank after fermentation ends. In the present invention, the total volume of the fermentation tank equipment is 75 m 3 . In the examples and comparative examples of the present invention, fermentation tanks with a total volume of 75 m 3 are used.

[0115] As can be seen from Table 1, in Examples 1 and 2, the change in fermentation pH reflects the growth of the bacterial cells. By feedback regulating the glucose flow rate based on the change in pH and simultaneously feeding a nutrient solution containing a fermentation intensifier, the fermentation process can be precisely controlled. When the fermentation reaches a certain degree, L-isoleucine is timely separated through a fermentation auxiliary device, avoiding the feedback inhibition caused by product accumulation and preventing the crystallization of L-isoleucine from clogging the pipeline and forcing the fermentation to stop. The separated nutrients and bacterial cells are returned to the fermenter for continued fermentation culture, improving the fermentation yield, equipment utilization rate, and fermentation production intensity.

[0116] In Comparative Example 1, a glucose solution was added at a constant rate. During the control process, the sugar concentration may be too high or too low. Being too high will inhibit the growth of the bacterial cells, and being too low will affect the metabolism of the bacterial cells and further affect the production and accumulation of L-isoleucine.

[0117] In Comparative Example 2, a nutrient solution was added at a constant rate. During the control process, there may be a situation of excessive or insufficient nutrients. Being too high will cause the bacterial cells to multiply abundantly, resulting in a low conversion rate. Being too low will inhibit the growth of the bacterial cells, and the bacterial cells will decline rapidly with a low acid production.

[0118] In Comparative Example 3, no fermentation intensifier was added, which would cause a low metabolic enzyme activity, slow metabolism, an extended fermentation cycle, and a low conversion rate.

[0119] In Comparative Example 4, although the fermentation process was precisely controlled, no fermentation auxiliary device was used. When the fermentation cycle reached 31 h, a large amount of crystals appeared in the fermenter, clogging the pipeline and affecting the fermentation, and the fermentation had to be stopped halfway.

[0120] In Comparative Example 5, according to the traditional fermentation method, the fermentation process cannot be precisely controlled, and no fermentation auxiliary device is used. Eventually, both the yield and conversion rate are low, the equipment utilization rate is low, and the production cost increases.

[0121] Comparative Example 6

[0122] Same as Example 1, the only difference is that cetyl sulfobetaine is not added to the fermentation intensifier. The fermentation indexes are shown in Table 2.

[0123] Comparative Example 7

[0124] Same as Example 1, the only difference is that octadecyl dimethyl betaine is not added to the fermentation intensifier. The fermentation indexes are shown in Table 2.

[0125] Comparative Example 8

[0126] Same as Example 1, the only difference is that CGF is not added to the fermentation intensifier. The fermentation indexes are shown in Table 2.

[0127] Comparative Example 9

[0128] Same as Example 1, the only difference is that cobalt chloride is not added to the fermentation intensifier. The fermentation indexes are shown in Table 2.

[0129] Comparative Example 10

[0130] Same as Example 1, the only difference is that copper chloride is not added to the fermentation intensifier. The fermentation indexes are shown in Table 2.

[0131] Table 2 Fermentation index data in Comparative Examples 6 - 10

[0132]

[0133] The fermentation index data of Comparative Examples 6 - 10 are shown in Table 2, and it can be seen that:

[0134] In Comparative Examples 6 - 10, when a certain component is lacking in the fermentation intensifier, the fermentation cycle is prolonged by 3 - 4 h compared with the Example, and both the fermentation production intensity and the conversion rate decrease. It shows that this fermentation intensifier is the best one suitable for the fermentation production of L - isoleucine, and the lack of any one component has a great impact on the fermentation production.

[0135] Thus, the technical solution in the present invention has the following advantages: (1) Under the combined action of the fermentation intensifier cetyl sulfobetaine and octadecyl dimethyl betaine surfactants, cobalt chloride and copper chloride metal ions, and CGF nutrients, the cell membrane permeability of the L - isoleucine - fermenting bacteria is better, nutrients can quickly enter the cells, and the metabolic activity of the bacteria is greatly enhanced, improving the fermentation production efficiency of L - isoleucine; (2) The regulation rate of pH directly reflects the growth rate of microorganisms. By feedback - regulating the glucose flow rate through the change of pH and simultaneously feeding a nutrient solution containing the fermentation intensifier, the L - isoleucine fermentation process is precisely controlled, enabling the bacteria to rapidly accumulate and produce L - isoleucine, shortening the fermentation cycle, and increasing the fermentation conversion rate; (3) Using the fermentation production auxiliary device, L - isoleucine in the fermentation broth is quickly crystallized and separated from the fermentation broth, and nutrients and bacteria etc. re - enter the fermentation tank for continued fermentation culture, avoiding the feedback inhibition caused by the accumulation of products and the forced stop of fermentation due to the blockage of the pipeline by L - isoleucine crystals, thereby achieving an increase in fermentation yield, an increase in the utilization rate of the fermentation tank, and production efficiency.

[0136] Although the above - mentioned embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for improving the fermentation production efficiency of L-isoleucine, characterized in that: The steps include: Corynebacterium glutamicum is inoculated into a fermentation medium for fermentation culture. When the glucose concentration in the fermentation liquid is less than 0.5% (w / v), a glucose solution and a nutrient solution are started to be fed, wherein the nutrient solution contains a fermentation enhancer. During the fermentation culture, liquid ammonia is fed to maintain the pH value of the fermentation culture at 7.3-7.5, and the volume of the liquid ammonia fed is determined according to the change in the pH value. The concentration of the glucose solution is 50%-55% (w / v). The volume of the fed glucose solution is determined according to the volume of the liquid ammonia fed per unit time, and the volume ratio of the liquid ammonia fed to the fed glucose solution per unit time is 1:(15-16). The volume of the fed nutrient solution is determined according to the volume of the glucose solution fed per unit time, and the volume ratio of the fed glucose solution to the fed nutrient solution per unit time is (10-11):

1. The fermentation enhancer uses water as solvent, and the component concentrations are as follows: 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; 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; the separation method includes pressing the fermentation liquid into a separation device.

2. The method for improving the fermentation efficiency of L-isoleucine according to claim 1, characterized in that: The nutrient solution uses water as a solvent and includes the following components in the following concentrations: 8-10 g / L of the fermentation enhancer, 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.

3. The method for improving the fermentation production efficiency of L-isoleucine according to claim 1, 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.

4. The method for improving the fermentation production efficiency of L-isoleucine according to claim 1, 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.

5. The method for improving the fermentation production efficiency of L-isoleucine according to claim 1, 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.

6. The method for improving the fermentation efficiency of L-isoleucine according to claim 1, characterized in that: During the fermentation culture, L-isoleucine is separated 2 to 3 times in the fermentation broth.

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