Preparation method of L-valine

Abstract

The invention provides a preparation method of L-valine. The method comprises the following steps: (1) immobilizing thalli in a seed culture solution by adopting a hollow cellulose membrane reactor in a fermentation system to obtain thalli immobilized on a hollow cellulose membrane; wherein the hollow cellulose membrane reactor comprises a hollow cellulose membrane; (2) circularly flushing the thalli fixed on the hollow cellulose membrane by adopting a fermentation culture medium to ferment the thalli fixed on the hollow cellulose membrane, and performing temperature control on the fermentation process by utilizing a plate heat exchanger to obtain fermentation liquor; and (3) carrying out anaerobic catalysis on the fermentation liquor by utilizing a catalytic culture medium. The method has obvious energy-saving and consumption-reducing effects, does not need to filter again to remove bacteria and impurities, and reduces the cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation, in particular to a preparation method of L-valine. BACKGROUND

[0002] L-valine, also known as 2-amino-3-methylbutyric acid, is an essential amino acid, which has a wide range of applications in human nutritional additives, feed additives, condiments, medicines, pesticides and health products.

[0003] There are four main methods for preparing L-valine: protein hydrolysis, chemical synthesis, enzymatic method and biological fermentation method. Since the raw materials for biological fermentation method are easy to obtain, the production cost is simple, and it is easy to produce on a large scale, the existing technology generally uses this method to produce L-valine. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art, and provides a preparation method of L-valine. The method does not require a large fermentation tank, saves space, and can directly connect an ammonia water pipeline, pH, temperature electrode, dissolved oxygen electrode and other electrodes in the hollow cellulose membrane reactor, and control the fermentation process in real time to ensure that the pH, temperature and other indicators required in the fermentation process meet the technical requirements.

[0005] The present application provides a preparation method of L-valine, comprising the following steps:

[0006] (1) using a hollow cellulose membrane reactor to immobilize the bacteria in the seed culture solution to obtain bacteria immobilized on the hollow cellulose membrane; wherein the hollow cellulose membrane reactor comprises a hollow cellulose membrane;

[0007] (2) using a fermentation medium to circulate the bacteria immobilized on the hollow cellulose membrane, so that the bacteria immobilized on the hollow cellulose membrane are fermented, and a plate heat exchanger is used to control the temperature of the fermentation process to obtain a fermentation broth;

[0008] (3) using a catalytic medium to anaerobically catalyze the fermentation broth.

[0009] Compared with the plate heat exchanger used in the prior art to control the temperature of the fermentation process, the plate heat exchanger has higher heat exchange efficiency, smaller water consumption, and the advantage of saving energy.

[0010] In some embodiments, the hollow cellulose membrane reactor has a membrane shell and membrane tube structure. The structure is simple, the size is flexible, and it is easy to install and place.

[0011] In some embodiments, the hollow cellulose membrane in the hollow cellulose membrane module has a pore size of 20-80 nm, for example, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, or any value therebetween.

[0012] In some embodiments, the fermentation is carried out in a fermentation tank.

[0013] In some embodiments, the effective volume (capacity) of the hollow cellulose membrane reactor is about 70% of the volume (capacity) of the fermentation tank.

[0014] In some embodiments, the effective area of the hollow cellulose membrane in the hollow cellulose membrane module is 1 m 2 Corresponding to 20-50 L of fermentation liquid passing through per minute, for example, 100 m 3 The fermentation tank has an initial liquid volume of 50 m 3 , and the final volume is 70-80 m 3 , and the required membrane area is about 320 m 2 .

[0015] In some embodiments, in step (1), the cellulose membrane reactor inlet is connected to an ammonia water pipeline to control the pH during fermentation.

[0016] In some embodiments, the ammonia water pipeline is connected to ammonia water or liquid ammonia.

[0017] In some embodiments, in step (2), the pH during fermentation is controlled to be 7.2±0.1. When the pH value in the reactor is lower than the set value by 0.1-0.5, the opening time (or opening frequency) of the control valve of the ammonia water flow pipeline is extended to control the pH value in the reaction period to be the set value.

[0018] In some embodiments, the fermentation temperature is 30-40℃, for example 30℃, 32℃, 34℃, 36℃, 38℃, 40℃, or any value therebetween. According to the change of the temperature in the reactor, when the temperature in the reactor is higher than the set value by 0.5-2℃, the circulation flow rate of the fermentation broth is increased; otherwise, the circulation flow rate of the fermentation broth is decreased and the temperature of the plate heat exchanger is increased. During the biological fermentation reaction, biological heat is generated. In order to control the temperature in the reactor, when the temperature in the reactor is higher than the set value (30-40℃) by 0.5-2℃, the circulation flow rate of the fermentation broth is increased, because the heat exchange amount is increased by increasing the flow rate, thereby achieving the purpose of cooling. Otherwise, when the temperature in the reactor is lower than the set value, the circulation flow rate of the fermentation broth is decreased and the temperature of the plate heat exchanger is increased. This is because when the flow rate is decreased, the heat exchange amount is correspondingly decreased, the culture temperature is increased, and the temperature is properly adjusted by increasing the temperature of the plate heat exchanger, so as to maintain the temperature in the reactor within a suitable range and ensure the smooth progress of the biological fermentation process and the fermentation effect.

[0019] In some embodiments, the ventilation ratio of the fermentation is 1:(0.1-0.5), for example 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, or any value therebetween.

[0020] In some embodiments, according to the liquid volume, the circulation flow is controlled once every 10-20 minutes.

[0021] In some embodiments, in step (2), the fermentation medium comprises: glucose 50-150 g / L, ammonium sulfate 10-20 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.5-2 g / L, yeast extract powder 1-5 g / L, proteose peptone 0.5-2 g / L, ferrous sulfate 5-15 mg / L, manganese sulfate 5-10 mg / L, copper sulfate 1-5 mg / L, biotin 0.01-0.02 mg / L, vitamin B1 2-6 mg / L, and kanamycin 20-30 mg / L.

[0022] In some embodiments, the fermentation medium comprises: glucose 100 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, yeast extract powder 2 g / L, proteose peptone 1 g / L, ferrous sulfate 10 mg / L, manganese sulfate 6 mg / L, copper sulfate 2 mg / L, biotin 0.01 mg / L, vitamin B1 4 mg / L, and kanamycin 25 mg / L.

[0023] In some embodiments, the catalytic medium comprises: glucose 50-80 g / L, ammonium sulfate 5-15 g / L, potassium dihydrogen phosphate 0.5-2 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.5-2 g / L, ferrous sulfate 5-15 mg / L, manganese sulfate 1-5 mg / L, and copper sulfate 1-5 mg / L.

[0024] In some embodiments, the catalytic medium comprises: glucose 60 g / L, ammonium sulfate 10 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1.5 g / L, magnesium sulfate 0.6 g / L, ferrous sulfate 10 mg / L, manganese sulfate 3 mg / L, and copper sulfate 1.5 mg / L.

[0025] In some embodiments, when the OD 610 of the fermentation liquid is 30-50, anaerobic catalysis is performed. At this time, the residual sugar in the fermentation medium is generally about 10 g / L, the residual fermentation medium is discharged, and the sterilized catalytic medium is added to perform catalytic reaction.

[0026] In some embodiments, the anaerobic catalytic conditions are as follows:

[0027] When the residual sugar amount is <15 g / L, glucose is added to a sugar concentration of 65-80 g / L; and when the residual sugar amount is <5 g / L, the catalytic reaction is ended (fermentation is ended).

[0028] In some embodiments, the concentration of the glucose solution is 350-450 g / L, such as but not limited to 350 g / L, 400 g / L, 450 g / L, and any value therebetween.

[0029] In some embodiments, the concentration of the glucose solution is 400 g / L.

[0030] In some embodiments, the end point of the anaerobic catalysis is that the residual sugar amount is <5 g / L.

[0031] In some embodiments, the amount of the seed culture solution is 10-25% of the volume of the fermentation medium, such as 10%, 15%, 20%, 25%, or any value therebetween.

[0032] In some embodiments, the preparation method of the seed culture solution comprises: inoculating a bacterial strain into a seed culture medium until the seed is mature, to obtain the seed culture solution.

[0033] In some embodiments, the bacterial strain is selected from Escherichia coli and / or Corynebacterium glutamicum.

[0034] In some embodiments, the seed maturation criteria comprise: OD 600 of 16-20.

[0035] In some embodiments, the preparation of the seed culture solution is performed in a seed tank.

[0036] In some embodiments, the seed culture medium comprises: glucose 25-35 g / L, ammonium sulfate 10-20 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 0.1-2 g / L, magnesium sulfate 0.5-2 g / L, yeast extract 2-8 g / L, proteose peptone 1-5 g / L, ferrous sulfate 5-15 mg / L, manganese sulfate 2-8 mg / L, copper sulfate 2-8 mg / L, biotin 0.01-0.02 mg / L, kanamycin 15-25 mg / L.

[0037] In some embodiments, the seed culture medium comprises: glucose 30 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 1.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate 1 g / L, yeast extract 5 g / L, proteose peptone 2 g / L, ferrous sulfate 10 mg / L, manganese sulfate 5 mg / L, copper sulfate 5 mg / L, biotin 0.01 mg / L, kanamycin 20 mg / L.

[0038] In some embodiments, the culture conditions of the seed culture solution meet at least one of the following culture conditions:

[0039] In some embodiments, the pH is 7.2±0.1.

[0040] In some embodiments, the culture temperature of the seed culture solution is 30-40℃, for example, 30℃, 32℃, 34℃, 36℃, 38℃, 40℃, or any value therebetween.

[0041] In some embodiments, the ventilation ratio during the culture process of the seed culture solution is 1:(0.1-1), for example, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, or any value therebetween. In some embodiments, the ventilation ratio during the culture process of the seed culture solution is 1:0.5.

[0042] In some embodiments, the preparation method further comprises: (4) concentrating, crystallizing, and drying the fermentation solution after fermentation to obtain the finished product L-valine.

[0043] In some embodiments, the fermentation period in the preparation method of the present application is 45-55 h.

[0044] In some embodiments, the acid production in the preparation method of the present application is 110-125 g / L.

[0045] In some embodiments, the sugar acid conversion rate in the preparation method of the present application is 40-50%.

[0046] Compared with the traditional fermentation process, the present application has the following beneficial effects:

[0047] (1) The present application does not need a large fermentation tank, saves space, can directly access ammonia water pipeline, feeding pipeline, and pH, temperature electrode, dissolved oxygen electrode, etc. in the cellulose membrane reactor, controls the fermentation process in real time, and quantitatively supplements nutrients and inducers through the feeding pipeline, to ensure that the pH, temperature, dissolved oxygen and other indicators required in the fermentation process meet the technical requirements;

[0048] (2) At the same time, since the cellulose membrane reactor is used to retain and fix the bacteria, the fermentation medium reaction is no longer needed to be filtered to remove bacteria and impurities after the reaction, and can be directly entered into the concentration process, simplifying the process and reducing the cost;

[0049] (3) The heat exchange efficiency of the plate heat exchanger is much higher than that of the coil and tube heat exchange mode of the traditional fermentation tank, and the energy saving and consumption reduction effect is obvious. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The L-valine production flowchart of an embodiment of the present application. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the following will further describe the present application in combination with examples and drawings. The specific examples described herein are only used to explain the present application and do not constitute any limitation on the present application.

[0052] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The endpoints of the ranges and the values are approximate values and should be understood to include values approximately near these ranges and values within these ranges. For ranges of values, the endpoints of the ranges are combined with the individual points to form new ranges of values that are within the scope of the present disclosure.

[0053] Unless defined, the technical terms used in the following examples have the same meaning as generally understood by those skilled in the art to which the present application belongs. The reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the raw materials, instruments and equipment used in the following examples can be purchased or obtained by existing methods; the amount of the reagents, unless otherwise specified, is the amount of the reagents in conventional experimental operation; the experimental methods, unless otherwise specified, are conventional methods.

[0054] The L-valine seed tank culture medium used in the present application is: glucose 30 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 1.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate 1 g / L, yeast extract powder 5 g / L, proteose peptone 2 g / L, ferrous sulfate 10 mg / L, manganese sulfate 5 mg / L, copper sulfate 5 mg / L, biotin 0.01 mg / L, kanamycin 20 mg / L, and ammonia water is used to control pH 7.2±0.1, the culture temperature is 32℃, the ventilation volume is 1:0.6, when OD 610 reaches 16-20, the seed maturation standard is reached.

[0055] The L-valine fermentation medium is: glucose 100 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, yeast extract powder 2 g / L, proteose peptone 1 g / L, ferrous sulfate 10 mg / L, manganese sulfate 6 mg / L, copper sulfate 2 mg / L, biotin 0.01 mg / L, vitamin B1 4 mg / L, kanamycin 25 mg / L, and ammonia water (or liquid ammonia) is used to control pH 7.2±0.1, the culture temperature is 32-35℃, the ventilation ratio is 1:03, when OD 610 reaches 30-50, stop aeration and enter the anaerobic catalysis stage.

[0056] The L-valine catalysis medium is: glucose 60 g / L, ammonium sulfate 10 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1.5 g / L, magnesium sulfate 0.6 g / L, ferrous sulfate 10 mg / L, manganese sulfate 3 mg / L, copper sulfate 1.5 mg / L, and ammonia water (or liquid ammonia) is used to control pH 7.2±0.1. The culture temperature is 35℃. Control the initial residual sugar to be 60 g / L, when the residual sugar is <15 g / L, add glucose to 70 g / L; when the residual sugar is <5 g / L, the fermentation is ended.

[0057] The ventilation ratio refers to the volume of air (L or m 3 ) per minute into the tank per volume (L or m 3 ) of the liquid in the tank.

[0058] OD 610 refers to the absorbance value of the seed culture solution and / or fermentation solution at a wavelength of 610 nm.

[0059] Example 1

[0060] According to the present application, the seed culture medium and the fermentation medium are optimized, and the ventilation ratio and the pH value are controlled, so that the yield of L-valine is increased, and the production cost is reduced. Figure 1The process described uses a 10L seed tank. 3L of the seed culture, having reached maturity under the aforementioned culture conditions, is passed through a pre-sterilized, sealed pipeline into a hollow cellulose membrane reactor within the fermentation system. The bacteria are filtered and attached to the hollow cellulose membrane (equivalent to immobilization), while the seed culture medium is filtered out. After completion, 30L of the prepared sterile fermentation medium, ten times the volume of the seed culture (30L of fermentation broth), is injected into the membrane reactor through a sterile pipeline. The medium circulates through the hollow cellulose membrane at a controlled flow rate, with each circulation cycle lasting 10-20 minutes. A plate heat exchanger is connected to this circulation system to control the fermentation temperature. Based on temperature changes within the reactor, when the reactor temperature exceeds the set value (33℃) by 1℃, the fermentation broth circulation rate is increased; conversely, when the temperature decreases, the circulation rate is decreased and the plate heat exchanger temperature is increased. An ammonia water pipeline is connected to the reactor inlet. When the pH value inside the reactor is 0.2 lower than the set value (7.5), i.e., the pH is 7.3, the opening time (or opening frequency) of the ammonia water flow control valve is extended to control the pH value inside the reactor to the set value. (Since the acid production stage of L-valine fermentation is an anaerobic stage, it is not necessary to introduce air into the reactor; therefore, only a stable culture medium is required.)

[0061] When OD is detected inside the reactor 610 When the concentration reaches 30-50%, the residual sugar in the fermentation medium is generally around 10 g / L. The remaining fermentation medium is then removed, and sterilized catalytic medium is added to initiate the catalytic reaction. When the residual sugar in the reactor is less than 15 g / L, 400 g / L glucose solution is added until the residual sugar reaches 65-80 g / L, and the reaction continues. When the residual sugar in the reactor is detected to be less than 5 g / L, the fermentation endpoint is reached. The fermentation cycle is 53.2 hours, with a final acid production of 110.46 g / L and a sugar-acid conversion rate of 42.76%, as shown in Table 1.

[0062] Example 2

[0063] Using 10 L seed tank, 6 L seed liquid which reached the maturation standard under the culture condition in Example 1, through the hollow cellulose membrane reactor in the fermentation system, the bacteria were filtered and attached to the hollow cellulose membrane (equivalent to immobilization), and the seed liquid medium was filtered out. After completion, the prepared sterile fermentation medium was injected into the membrane reactor through the sterile pipeline at a dosage of 5 times the volume of the seed liquid, i.e. 30 L fermentation liquid, so that the medium circulated through the hollow cellulose membrane in the reactor at a certain flow rate. The circulation time was controlled at 10-20 min. At the same time, a plate heat exchanger was connected to the circulation system to control the temperature of the fermentation process. According to the change of the temperature in the reactor, when the temperature in the reactor was higher than the set value (33°C) by 0.5°C, the circulation flow rate of the fermentation liquid was increased, and vice versa. The temperature of the plate heat exchanger was increased. An ammonia water pipeline was connected to the inlet of the reactor, and when the pH value in the reactor was lower than the set value (7.5) by 0.1, i.e. pH 7.4, the opening time (or opening frequency) of the ammonia water flow adding pipeline control valve was extended, and the pH value in the reactor was controlled at the set value. (Since the L-valine fermentation acid production stage is an anaerobic stage, there is no need to supply air to the reactor, so only stable medium is needed.)

[0064] When the OD 610 When the residual sugar in the fermentation medium reached 30-50 g / L, the residual fermentation medium was discharged, the sterilized catalytic medium was added, and the catalytic reaction was carried out; when the residual sugar in the reactor was equal to 1.8 g / L, 400 g / L of glucose solution was supplemented to make the residual sugar reach 65-80 g / L, and the reaction was continued; when the residual sugar in the reactor was less than 5 g / L, the fermentation period was 46.5 h, and the final acid production was 119.45 g / L, and the sugar acid conversion rate was 46.24%.

[0065] Example 3

[0066] Using 500 L seed tank, 300 L seed liquid which reached the maturation standard under the culture condition in Example 1, through the hollow cellulose membrane reactor in the fermentation system, the bacteria were filtered and attached to the membrane (equivalent to immobilization), and the seed liquid medium was filtered out. After completion, the prepared sterile fermentation medium was injected into the membrane reactor through the sterile pipeline at a dosage of 5 times the volume of the seed liquid, i.e. 1500 L fermentation liquid, so that the medium circulated through the hollow cellulose membrane in the reactor at a certain flow rate. The circulation time was controlled at 10-20 min. At the same time, a plate heat exchanger was connected to the circulation system to control the temperature of the fermentation process. According to the change of the temperature in the reactor, when the temperature in the reactor is higher than the set value (33°C) by 0.5°C, the circulation flow rate of the fermentation liquid is increased, and vice versa. The ammonia pipeline is connected to the inlet of the reactor, and pH electrodes are installed at the inlet and outlet of the reactor. When the pH value in the reactor is lower than the set value (7.5) by 0.1, the opening time (or opening frequency) of the ammonia flow valve is extended; at the same time, when the difference between the values of the inlet and outlet pH electrodes is greater than 0.1, the flow rate of the fermentation liquid circulation system is also increased to control the pH value in the reaction period to be the set value. (Since the L-valine fermentation acid production stage is an anaerobic stage, there is no need to supply air to the reactor, so only a stable medium is needed.)

[0067] When the OD610 in the reactor is detected to be 30-50, at this time the residual sugar in the fermentation medium is generally about 10 g / L, the residual fermentation medium is discharged, the sterilized catalytic medium is added, and the catalytic reaction is carried out; when the residual sugar in the reactor is equal to 1.9 g / L, 400 g / L of glucose solution is supplemented to make the residual sugar reach 65-80 g / L, and the reaction is continued; when the residual sugar in the reactor is detected to be less than 5 g / L, the fermentation endpoint is reached. The fermentation period is 48.2 h, the final acid production is 120.78 g / L, and the sugar acid conversion rate is 45.43%.

[0068] Example 4

[0069] Using 20 m 3 L seed tank, 15 m 3 L seed liquid which reached the maturation standard under the culture condition in Example 1, through the hollow cellulose membrane reactor, the bacteria were filtered and attached to the membrane (equivalent to immobilization), and the seed liquid medium was filtered out. After completion, the prepared sterile fermentation medium was injected into the membrane reactor through the sterile pipeline at a dosage of 5 times the volume of the seed liquid, i.e. 75 m 3The fermentation broth is injected into the membrane reactor through a sterile pipeline, so that the medium circulates in the reactor at a certain flow rate through the hollow cellulose membrane, and a plate heat exchanger is connected in the circulation system to control the temperature of the fermentation process. According to the change of the temperature in the reactor, when the temperature in the reactor is higher than the set value (33°C) by 0.5°C, the circulation flow rate of the fermentation broth is increased, and vice versa, and the temperature of the plate heat exchanger is increased. A liquid ammonia pipeline is connected at the inlet of the reactor, and pH electrodes are installed at the inlet and outlet of the reactor. When the pH value in the reactor is lower than the set value (7.5) by 0.1, the opening time (or opening frequency) of the liquid ammonia flow pipeline control valve is extended; at the same time, when the difference between the values of the inlet and outlet pH electrodes is greater than 0.1, the circulation flow rate of the fermentation broth is also increased, and the pH value in the reaction period is controlled to be the set value. (Since the L-valine fermentation acid production stage is an anaerobic stage, there is no need to introduce air into the reactor, so only a stable medium needs to be provided.)

[0070] When the OD 610 When the residual sugar in the fermentation medium reaches 30-50, which is generally about 10 g / L, the residual fermentation medium is discharged, the sterilized catalytic medium is added, and the catalytic reaction is carried out; when the residual sugar in the reactor is equal to 1.6 g / L, 400 g / L of glucose solution is supplemented to make the residual sugar reach 65-80 g / L, and the reaction is continued; when the residual sugar in the reactor is detected to be less than 5 g / L, the fermentation endpoint is reached. The fermentation period is 47.5 h, the final acid production is 123.41 g / L, and the sugar acid conversion rate is 46.72%.

[0071] Comparative Example 1

[0072] The L-valine seed tank medium used in the 5L seed tank is as follows: glucose 30 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 1.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate 1 g / L, yeast extract powder 5 g / L, peptone 2 g / L, ferrous sulfate 10 mg / L, manganese sulfate 5 mg / L, copper sulfate 5 mg / L, biotin 0.01 mg / L, and kanamycin 20 mg / L. The pH is controlled at 7.1-7.2 by using ammonia water, the culture temperature is 32°C, and the ventilation volume is 1:0.5. When the OD 610When the temperature reaches 16-20°C, the seed matures. The resulting 3L of seed solution is inoculated into a 50L fermenter at 10% of the inoculum volume. The fermenter has a volume of 30L. The fermentation medium consists of: glucose 100g / L, ammonium sulfate 15g / L, potassium dihydrogen phosphate 2g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate 1g / L, yeast extract 2g / L, peptone 1g / L, ferrous sulfate 10mg / L, manganese sulfate 6mg / L, copper sulfate 2mg / L, biotin 0.01mg / L, vitamin B1 4mg / L, and kanamycin 25mg / L. Ammonia is used to control the pH at 7.1-7.2, and the culture temperature is 34°C. Before shutting down the fermenter, the ventilation ratio is 1:0.5. When the fermenter's OD... 610 When the pH reaches 30-50, the residual sugar in the fermentation medium is generally around 10 g / L. The fermentation broth is drained and transferred to a sterile centrifuge cup. It is then centrifuged at 4°C and 4000 rpm for 20 minutes, and the supernatant is discarded. The bacterial sludge is resuspended in sterilized catalytic medium (60 g / L glucose, 10 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 1.5 g / L dipotassium hydrogen phosphate, 0.6 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 3 mg / L manganese sulfate, and 1.5 mg / L copper sulfate) and transferred back to the fermenter. When the residual sugar in the fermenter reaches 2.0 g / L, 400 g / L glucose solution is added until the residual sugar reaches 65-80 g / L, and the reaction continues. When the residual sugar in the fermenter is detected to be less than 5 g / L, the fermentation endpoint is reached (the fermenter is not aerated during the catalytic stage). The fermentation cycle was 46.5 hours, with a final acid production of 116.46 g / L and a sugar-acid conversion rate of 44.61%.

[0073] Comparative Example 2:

[0074] 10m 3 The seed tank also uses the L-valine seed tank culture medium: glucose 30 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 1.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate 1 g / L, yeast extract 5 g / L, peptone 2 g / L, ferrous sulfate 10 mg / L, manganese sulfate 5 mg / L, copper sulfate 5 mg / L, biotin 0.01 mg / L, kanamycin 20 mg / L. Ammonia water is used to control the pH at 7.1-7.2. The culture temperature is 32℃, and the aeration rate is 1:0.5. When OD... 610 When the temperature reaches 16-20°C, the seed meets the maturity standard. The resulting 7m... 3 Inoculate 10% of the seed solution into 100m³ of water. 3 Fermentation tank. The fermentation tank has a volume of 70m³. 3, fermentation medium: glucose 100 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 2 g / L, potassium hydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, yeast extract 2 g / L, proteose peptone 1 g / L, ferrous sulfate 10 mg / L, manganese sulfate 6 mg / L, copper sulfate 2 mg / L, biotin 0.01 mg / L, vitamin B1 4 mg / L, kanamycin 25 mg / L, using ammonia water to control pH 7.1-7.2, culture temperature 34°C. Before the fermentation tank stops aeration, the ventilation volume is 1:0.5, when the OD 610 When the OD value of the fermentation tank reaches 30-50, at this time the residual sugar in the fermentation medium is generally about 10 g / L, the fermentation broth is added into a sterilized high-speed centrifuge by a pipeline. Freeze centrifugation, 4°C, 4000 rpm, centrifugation for 20 min, then the supernatant is discharged. The bacterial sludge is suspended with a sterilized catalytic medium (glucose 60 g / L, ammonium sulfate 10 g / L, potassium dihydrogen phosphate 1 g / L, potassium hydrogen phosphate 1.5 g / L, magnesium sulfate 0.6 g / L, ferrous sulfate 10 mg / L, manganese sulfate 3 mg / L, copper sulfate 1.5 mg / L), and is returned to the fermentation tank. When the residual sugar in the fermentation tank is equal to 1.6 g / L, 400 g / L of glucose solution is supplemented to make the residual sugar reach 65-80 g / L, and the reaction is continued; when it is detected that the residual sugar in the fermentation tank is less than 5 g / L, the fermentation endpoint is reached (the fermentation tank is not aerated in the catalytic stage). The fermentation period is 48 h, the final acid yield is 119.46 g / L, and the sugar acid conversion rate is 45.83%.

[0075] Table 1

[0076]

[0077] As can be seen from the examples and comparative examples, too small seed tank culture volume has a greater impact on the fermentation index, when the seed liquid volume reaches 20% of the fermentation volume (i.e. example 2, example 4), the fermentation index is obviously improved. Similarly, the control accuracy of the pH value in the reactor has a greater impact on the fermentation index, when the mixing effect is increased by increasing the flow rate, the control accuracy is improved by using a double electrode, and the like, the fermentation index is obviously improved. Finally, in a 100 m 3 In the fermentation tank, the production index using the hollow cellulose membrane reactor is basically the same as that of the conventional fermentation.

[0078] 100 m 3 The power of the stirring motor in the fermentation tank generally reaches 100 KW, and according to the operation load of 60-70%, the power is about 60-70 KW; while the hollow cellulose membrane reactor is used in the 100 m 3 The centrifugal pump (flow 300 m 3The power of the pump (head 30m) is about 45KW, and the power is about 31.5-36KW according to the operation load of 70-80%. Since the fermentation system of the present application does not need centrifugal pump and other devices, the total power of the fermentation tank in the prior art is 1200-1900KW different from the total power of the present application, calculated at 50h under the condition of similar fermentation cycle.

[0079] In addition, the hollow cellulose membrane reactor of the present application has simple structure, and does not need heat exchange tube, baffle, air distributor, stirring shaft, stirring paddle and other accessories, greatly reducing the bacteria contamination dead angle and the probability of bacteria contamination in the fermentation process. Meanwhile, the present application has low requirements for the shape structure of the storage tank and the placement position selection. After fermentation, since the bacteria are immobilized on the hollow cellulose membrane, the process of filtering the fermentation broth through the membrane to remove bacterial impurities is reduced, the labor is reduced, and the cost is saved.

[0080] In summary, the method for producing L-valine by fermentation of the present application effectively reduces the production cost without affecting the fermentation index. In the case of similar fermentation index, the energy consumption (including power consumption and circulating water consumption) is greatly reduced. Meanwhile, due to the simple structure of the reactor, the maintenance cost is reduced, and the probability of bacteria contamination is reduced. The plate heat exchanger is used to adjust the temperature, the heat exchange efficiency is high, and the circulating water consumption is significantly reduced.

[0081] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application, and belong to the protection scope of the present application.

Claims

1. A method for preparing L-valine, comprising the following steps: (1) using a hollow cellulose membrane reactor to immobilize the bacteria in the seed culture solution, to obtain bacteria immobilized on the hollow cellulose membrane; wherein, The hollow cellulose membrane reactor comprises a hollow cellulose membrane; (2) The bacteria fixed on the hollow cellulose membrane are cyclically flushed with a fermentation medium, so that the bacteria fixed on the hollow cellulose membrane are fermented, and a plate heat exchanger is used to control the temperature of the fermentation process, thereby obtaining a fermentation liquor; (3) The fermentation liquor is subjected to anaerobic catalysis with a catalytic medium.

2. The production method according to claim 1, characterized by, In step (1), the cellulose membrane reactor inlet is connected with an ammonia water pipeline to control the pH in the fermentation process; and the ammonia water pipeline is connected with ammonia water or liquid ammonia.

3. The preparation method according to claim 1, characterized in that, In step (2), the pH in the fermentation process is controlled to be 7.2±0.1; and / or In step (2), the fermentation temperature is 30-40℃; and / or In step (2), the ventilation ratio of the fermentation is 1:(0.1-0.5).

4. The production method according to claim 1, characterized by, In step (2), the fermentation medium comprises: glucose 50-150 g / L, ammonium sulfate 10-20 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.5-2 g / L, yeast extract powder 1-5 g / L, protein peptone 0.5-2 g / L, ferrous sulfate 5-15 mg / L, manganese sulfate 5-10 mg / L, copper sulfate 1-5 mg / L, biotin 0.01-0.02 mg / L, vitamin B12-6 mg / L, and kanamycin 20-30 mg / L. Preferably, in step (2), the fermentation medium comprises: glucose 100 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, yeast extract powder 2 g / L, protein peptone 1 g / L, ferrous sulfate 10 mg / L, manganese sulfate 6 mg / L, copper sulfate 2 mg / L, biotin 0.01 mg / L, vitamin B14 mg / L, and kanamycin 25 mg / L.

5. The preparation method according to claim 1, characterized in that, In step (3), the catalytic medium comprises: glucose 50-80 g / L, ammonium sulfate 5-15 g / L, potassium dihydrogen phosphate 0.5-2 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.5-2 g / L, ferrous sulfate 5-15 mg / L, manganese sulfate 1-5 mg / L, and copper sulfate 1-5 mg / L. Preferably, in step (3), the catalytic medium comprises: glucose 60 g / L, ammonium sulfate 10 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1.5 g / L, magnesium sulfate 0.6 g / L, ferrous sulfate 10 mg / L, manganese sulfate 3 mg / L, and copper sulfate 1.5 mg / L.

6. The method of claim 1, wherein, In step (2), when the OD of the fermentation broth... 610 When the concentration is 30-50, anaerobic catalysis begins; The anaerobic catalysis is performed under the following conditions: When the residual sugar amount is less than 15 g / L, the nutrition source glucose is added to a sugar concentration of 65-80 g / L; and when the residual sugar amount is less than 5 g / L, the anaerobic catalysis reaction is ended. Preferably, the nutrition source is a 350-450 g / L glucose solution, and most preferably a 400 g / L glucose solution.

7. The preparation method according to claim 1, characterized in that, The seed culture solution is used in an amount of 10-25% of the volume of the fermentation medium.

8. The production method according to claim 1 or 7, characterized by, The seed culture solution is prepared by inoculating bacteria into a seed culture medium until the seed matures. The seed culture solution is prepared by inoculating bacteria into a seed culture medium until the seed matures. Preferably, the bacterial strain is selected from Escherichia coli and / or Corynebacterium glutamicum. Seed maturity criteria include: OD 610 is 16-20; Preferably, the preparation of the seed culture solution is performed in a seed tank.

9. The production method according to claim 8, characterized by, The seed culture medium comprises: glucose 25-35 g / L, ammonium sulfate 10-20 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 0.1-2 g / L, magnesium sulfate 0.5-2 g / L, yeast extract 2-8 g / L, proteose peptone 1-5 g / L, ferrous sulfate 5-15 mg / L, manganese sulfate 2-8 mg / L, copper sulfate 2-8 mg / L, biotin 0.01-0.02 mg / L, kanamycin 15-25 mg / L. Preferably, the seed culture medium comprises: glucose 30 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 1.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate 1 g / L, yeast extract 5 g / L, proteose peptone 2 g / L, ferrous sulfate 10 mg / L, manganese sulfate 5 mg / L, copper sulfate 5 mg / L, biotin 0.01 mg / L, kanamycin 20 mg / L.

10. The preparation method according to claim 8, characterized in that, The culture conditions of the seed culture solution satisfy at least one of the following culture conditions: pH is 7.2±0.1, and / or The culture temperature of the seed culture solution is 30-40℃, and / or The ventilation ratio of the culture process of the seed culture solution is 1:(0.1-1), preferably 1:0.5.

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