A method for producing valine by fermentation and a fermentation device for producing valine

Abstract

The present application belongs to the technical field of valine fermentation, and particularly relates to a method for fermentatively producing valine and a fermentation device for producing valine. The present application provides a method for fermentatively producing valine, which utilizes fermentation tanks with different height-diameter ratios to fermentatively produce valine. A fermentation tank with a large height-diameter ratio is used when the bacteria grow, and a fermentation tank with a small height-diameter ratio is used when valine is produced. Meanwhile, the low-oxygen-content tail gas of the first fermentation culture (aerobic fermentation) is used to provide air for the second fermentation culture (anaerobic fermentation), thus realizing semi-continuous fermentation of valine, saving the processes of primary and secondary seed expansion and sterilization and cleaning of the seed tank, greatly improving the equipment utilization rate, shortening the fermentation production chain, reducing the probability of contamination, and shortening the fermentation period. The method provided by the present application has low requirements for personnel during the fermentation process, the fermentation process control is more accurate, air is saved and energy is conserved, the conversion rate of valine and the utilization rate of the fermentation tank are improved, and the fermentation efficiency is high.

Description

Technical Field

[0001] The invention belongs to the technical field of valine fermentation, and particularly relates to a method for producing valine by fermentation and a fermentation device for producing valine. Background Art

[0002] Valine is an essential amino acid for the human body. It promotes growth, repairs tissues, regulates blood sugar, and provides energy. It also aids in the recovery of patients with liver disease and repairs liver damage caused by alcoholism and drug abuse. Valine is widely used in food, medicine, cosmetics, and animal feed. It is used as a food additive, feed additive, nutritional supplement, and a precursor to drug preparation, and its market prospects are promising.

[0003] Currently, the primary method for producing valine is fermentation. After primary seed culture and secondary seed expansion, the valine-producing strain undergoes batch fermentation to produce a valine-rich fermentation broth, which is then extracted to produce the valine product. However, this method is time-consuming and has a long fermentation process. The seed culture process carries a high risk of contamination, and the production and fermentation control process is complex and requires high technical expertise. Fermentation control conditions must be constantly adjusted based on the strain's production status. Summary of the Invention

[0004] The object of the present invention is to provide a method for producing valine by fermentation and a fermentation device for producing valine, which have high valine fermentation efficiency and short production chain and production cycle.

[0005] The present invention provides a method for producing valine by fermentation, comprising the following steps:

[0006] (1) adding 10% to 15% of the volume of the first fermentation tank to the first fermentation tank, inoculating the seed liquid and starting the first fermentation culture to obtain the first fermentation liquid;

[0007] The height-to-diameter ratio of the first fermentation tank is (4-6):1;

[0008] The seed liquid is the seed liquid of a valine-producing strain;

[0009] During the first fermentation culture, after the initial sugar in the fermentation medium is exhausted, nutrients are added to make the glucose concentration in the fermentation broth 0.5-0.8 wt.%;

[0010] (2) When the OD of the first fermentation liquid in step (1) 610nmWhen the value is ≥0.8 and the volume of the first fermentation broth is ≥75% of the volume of the first fermentation tank, 60%-65% of the first fermentation broth is transferred to a second fermentation tank for a second fermentation culture to obtain a valine fermentation broth; the height-to-diameter ratio of the second fermentation tank is (2-4):1; during the second fermentation culture, the tail wind of the first fermentation tank is used as an air source, and a fermentation feed is fed to ensure that the glucose concentration of the fermentation broth in the second fermentation tank is 0.2-0.3 wt.%;

[0011] After 60% to 65% of the first fermentation liquid is transferred to the first fermentation tank, the nutrients in step (1) are added to the first fermentation tank to continue fermentation and cultivation; during the continued fermentation and cultivation, the glucose concentration in the fermentation liquid in the first fermentation tank is maintained at 0.5 to 0.8 wt.%, until the OD value of the fermentation liquid in the first fermentation tank reaches 0.5 to 0.8 wt.%. 610nm The value is ≥0.8, and the volume of the fermentation liquid is ≥75% of the volume of the first fermentation tank;

[0012] (3) Extracting the valine fermentation broth described in step (2) to obtain valine.

[0013] Preferably, the temperature of the first fermentation culture is 35-37°C, the stirring frequency is 35-60 r / min, and the dissolved oxygen content of the fermentation liquid is maintained at 15%-30%;

[0014] The temperature of the continued fermentation culture is 35-37°C, the stirring frequency is 35-60 r / min, and the dissolved oxygen content of the fermentation liquid is maintained at 15%-30%;

[0015] The temperature of the second fermentation culture is 35-38° C., the stirring frequency is 15-30 r / min, and the dissolved oxygen content of the fermentation liquid is maintained below 5%.

[0016] Preferably, the fermentation medium in step (1) and step (2) respectively comprises the following components in the following concentrations: yeast powder 1-3 g / L, peptone 1-2 g / L, corn steep liquor 5-7 g / L, betaine 1-1.2 g / L, glucose 10-15 g / L, magnesium sulfate 0.5-0.8 g / L, potassium dihydrogen phosphate 1.5-2 g / L, biotin 3-6 mg / L and calcium pantothenate 20-25 mg / L.

[0017] Preferably, the nutrients in step (1) and step (2) respectively include components with the following concentrations: yeast powder 0.5-1 g / L, peptone 1-1.5 g / L, corn steep liquor 3-5 g / L, betaine 2-3 g / L, glucose 70-75 g / L, magnesium sulfate 0.5-0.8 g / L and potassium dihydrogen phosphate 0.8-1 g / L.

[0018] Preferably, the fermentation material in step (2) comprises the following components at the following concentrations: peptone 3-5 g / L, glucose 300-350 g / L, magnesium sulfate 1-2 g / L, potassium dihydrogen phosphate 1.5-2 g / L, biotin 12-15 mg / L, cobalt chloride 20-25 mg / L, copper sulfate 5-8 mg / L

[0019] Preferably, the extracting step comprises:

[0020] performing a first filtration on the valine fermentation broth using a ceramic membrane to collect a first filtrate;

[0021] performing a second filtration on the first filtrate using a nanofiltration membrane to collect the second filtrate;

[0022] sequentially concentrating, centrifuging and drying the second filtrate to obtain valine;

[0023] The ceramic membrane has a core thickness of 30-200 nm and a transmembrane pressure difference of ≤10.0 bar;

[0024] The membrane core of the nanofiltration membrane is 200~1000Dal, and the transmembrane pressure difference is 18~20bar.

[0025] Preferably, the concentration is evaporation concentration, and the output density of the evaporation concentration is 1.15-1.22 g / mL.

[0026] Preferably, the method further comprises: after obtaining the valine fermentation broth, collecting the valine fermentation broth, and repeating step (2) until the fermentation efficiency of the second fermentation tank decreases or the first fermentation tank is contaminated with bacteria.

[0027] Preferably, the number of repetitions is 30 to 80 times.

[0028] The present invention also provides a fermentation device for producing valine, comprising a first fermentation tank and a second fermentation tank; an exhaust port of the first fermentation tank is connected to an air inlet of the second fermentation tank through a pipeline;

[0029] The discharge port of the first fermentation tank is connected to the feed port of the second fermentation tank through a pipeline;

[0030] The height-to-diameter ratio of the first fermentation tank is (4-6):1; the height-to-diameter ratio of the second fermentation tank is (2-4):1.

[0031] Beneficial effects:

[0032] The present invention provides a method for producing valine by fermentation, comprising the following steps: (1) adding a fermentation medium of 10% to 15% of the volume of the first fermentation tank into a first fermentation tank, inoculating a seed liquid for a first fermentation culture, and obtaining a first fermentation liquid; the height-to-diameter ratio of the first fermentation tank is (4 to 6): 1; the seed liquid is a seed liquid of a strain producing valine; during the first fermentation culture, after the initial sugar in the fermentation medium is exhausted, nutrients are supplemented to make the glucose concentration in the fermentation liquid 0.5 to 0.8 wt.%; (2) when the OD of the first fermentation liquid in step (1) is 610nm When the value is ≥0.8 and the volume of the first fermentation liquid is ≥75% of the volume of the first fermentation tank, 60%~65% of the first fermentation liquid is transferred to the second fermentation tank for second fermentation culture to obtain valine fermentation liquid; the height-to-diameter ratio of the second fermentation tank is (2~4):1; during the second fermentation culture, the tail wind of the first fermentation tank is used as the air source, and the fermentation material is added to make the glucose concentration of the fermentation liquid in the second fermentation tank 0.2~0.3wt.%; after the first fermentation tank transfers 60%~65% of the first fermentation liquid, the nutrients described in step (1) are added to the first fermentation tank to continue fermentation culture; during the continued fermentation culture, the glucose concentration in the fermentation liquid in the first fermentation tank is maintained at 0.5~0.8wt.%, until the OD of the fermentation liquid in the first fermentation tank reaches 0. 610nm The value is ≥0.8, and the volume of the fermentation liquid is ≥75% of the volume of the first fermentation tank; (3) the valine fermentation liquid of step (2) is extracted to obtain valine. The present invention utilizes fermentation tanks with different height-to-diameter ratios to ferment and produce valine. An aerobic fermentation tank with a large height-to-diameter ratio is used when the bacteria grow, and an oxygen-limited fermentation tank with a small height-to-diameter ratio is used when producing valine. At the same time, the low-oxygen tail gas of the first fermentation culture (aerobic fermentation) is used to provide air for the second fermentation culture (anaerobic fermentation), thereby realizing semi-continuous fermentation of valine, saving the microbial primary and secondary seed expansion and seed tank sterilization and cleaning processes, greatly improving equipment utilization, greatly shortening the fermentation production chain, reducing the probability of bacterial contamination, and shortening the fermentation cycle. The method provided by the present invention has low personnel requirements for the fermentation process, more precise fermentation process control, gas and energy saving, improved valine conversion rate and fermentation tank utilization, and high fermentation efficiency. DETAILED DESCRIPTION

[0033] The present invention provides a method for producing valine by fermentation, comprising the following steps:

[0034] (1) adding 10% to 15% of the volume of the first fermentation tank to the first fermentation tank, inoculating the seed liquid to perform the first fermentation culture, and obtaining the first fermentation liquid;

[0035] The height-to-diameter ratio of the first fermentation tank is (4-6):1;

[0036] The seed liquid is the seed liquid of a valine-producing strain;

[0037] During the first fermentation culture, after the initial sugar in the fermentation medium is exhausted, nutrients are added to make the glucose concentration in the fermentation broth 0.5-0.8 wt.%;

[0038] (2) When the OD of the first fermentation liquid in step (1) 610nm When the value is ≥0.8 and the volume of the first fermentation broth is ≥75% of the volume of the first fermentation tank, 60%-65% of the first fermentation broth is transferred to a second fermentation tank for a second fermentation culture to obtain a valine fermentation broth; the height-to-diameter ratio of the second fermentation tank is (2-4):1; during the second fermentation culture, the tail wind of the first fermentation tank is used as an air source, and a fermentation feed is fed to ensure that the glucose concentration of the fermentation broth in the second fermentation tank is 0.2-0.3 wt.%;

[0039] After 60% to 65% of the first fermentation liquid is transferred to the first fermentation tank, the nutrients in step (1) are added to the first fermentation tank to continue fermentation and cultivation; during the continued fermentation and cultivation, the glucose concentration in the fermentation liquid in the first fermentation tank is maintained at 0.5 to 0.8 wt.%, until the OD value of the fermentation liquid in the first fermentation tank reaches 0.5 to 0.8 wt.%. 610nm The value is ≥0.8, and the volume of the fermentation liquid is ≥75% of the volume of the first fermentation tank;

[0040] (3) Extracting the valine fermentation broth described in step (2) to obtain valine.

[0041] The present invention adds 10% to 15% of the volume of the first fermentation tank to a fermentation medium, inoculates a seed solution, and then begins a first fermentation culture to obtain a first fermentation liquid. As an embodiment, the present invention adds 15% of the volume of the first fermentation tank to a fermentation medium, inoculates a seed solution, and then begins a first fermentation culture to obtain a first fermentation liquid.

[0042] In the present invention, the height-to-diameter ratio of the first fermenter is (4-6):1. As an embodiment, the height-to-diameter ratio of the first fermenter of the present invention is 4:1. As an embodiment, the first fermenter of the present invention includes a tank wall, a temperature-controlled internal coil, a feed port, a discharge port, an air inlet, an exhaust port, a stirring device, and a driving device. The feed port and the exhaust port are respectively arranged at the top of the fermenter cavity, the temperature-controlled internal coil is distributed around the fermenter cavity, the discharge port and the air inlet are arranged at the bottom of the fermenter cavity, the driving device is arranged at the top of the fermenter and is connected to the stirring device, the stirring device extends from the top of the fermenter to the bottom of the tank cavity, and the stirring device is provided with a stirring paddle. The present invention has no strict requirements on the source of the first fermenter, and conventional purchase is sufficient.

[0043] In the present invention, the seed liquid is the seed liquid of a strain producing valine. As an embodiment, the strain producing valine in the present invention is Escherichia coli. The present invention has no special restrictions on the specific source of the Escherichia coli, and any strain of Escherichia coli conventionally used in the art for fermentation and preparation of L-valine can be used. The present invention has no strict requirements on the preparation method of the seed liquid, and the strain producing valine can be fermented and cultured using a culture medium. The present invention has no special restrictions on the source of the components in the culture medium, and they can be purchased through conventional commercial channels.

[0044] In the present invention, during the first fermentation culture, after the initial sugar is depleted, nutrients are supplemented to bring the glucose concentration in the fermentation broth to 0.5-0.8 wt.%. In one embodiment, the nutrients of the present invention include the following components at the following concentrations: yeast powder 0.5-1 g / L, peptone 1-1.5 g / L, corn steep liquor 3-5 g / L, betaine 2-3 g / L, glucose 70-75 g / L, magnesium sulfate 0.5-0.8 g / L, and potassium dihydrogen phosphate 0.8-1 g / L; in another embodiment, the nutrients of the present invention include the following components at the following concentrations: yeast powder 0.5 g / L, peptone 13 g / L, corn steep liquor 4 g / L, betaine 3 g / L, glucose 75 g / L, magnesium sulfate 0.5 g / L, and potassium dihydrogen phosphate 0.8 g / L. In one embodiment, the solvent for the nutrients of the present invention is water.

[0045] In one embodiment, during the first fermentation culture of the present invention, after the initial sugar in the fermentation medium is depleted, nutrients are supplemented to achieve a glucose concentration in the fermentation broth of 0.6 wt.%. In one embodiment, the temperature of the first fermentation culture of the present invention is 35-37°C; in another embodiment, the temperature of the first fermentation culture of the present invention is 36.3-36.7°C; in another embodiment, the temperature of the first fermentation culture of the present invention is 36.5°C. In one embodiment, the stirring frequency of the first fermentation culture of the present invention is 35-60 r / min; in another embodiment, the stirring frequency of the first fermentation culture of the present invention is 50 r / min. In one embodiment, the dissolved oxygen content of the fermentation broth of the first fermentation culture of the present invention is maintained at 15%-30%; in another embodiment, the dissolved oxygen content of the fermentation broth of the first fermentation culture of the present invention is maintained at 20%. In one embodiment, the present invention maintains dissolved oxygen in the fermentation broth of the first fermentation culture by adjusting the ventilation ratio. In one embodiment, the ventilation ratio of the present invention is 0.25 to 1.0 L / (L·min); in another embodiment, the ventilation ratio of the present invention is 0.3 to 0.8 L / (L·min); in another embodiment, the ventilation ratio of the present invention is 0.5 L / (L·min). The first fermentation culture of the present invention is aerobic fermentation.

[0046] In one embodiment, the fermentation medium of the present invention comprises 1-3 g / L yeast extract, 1-2 g / L peptone, 5-7 g / L corn steep liquor, 1-1.2 g / L betaine, 10-15 g / L glucose, 0.5-0.8 g / L magnesium sulfate, 1.5-2 g / L potassium dihydrogen phosphate, 3-6 mg / L biotin, and 20-25 mg / L calcium pantothenate. In another embodiment, the fermentation medium of the present invention comprises 2.5 g / L yeast extract, 1.5 g / L peptone, 5 g / L corn steep liquor, 1.2 g / L betaine, 12 g / L glucose, 0.6 g / L magnesium sulfate, 2 g / L potassium dihydrogen phosphate, 3.8 mg / L biotin, and 20 mg / L calcium pantothenate. In one embodiment, the solvent of the fermentation medium of the present invention is water.

[0047] In the present invention, when the OD of the first fermentation broth is 610nm When the OD value of the first fermentation broth is ≥ 0.8 and the volume of the first fermentation broth is ≥ 75% of the volume of the first fermentation tank, 60-65% of the first fermentation broth is transferred to the second fermentation tank for the second fermentation to obtain the valine fermentation broth. 610nm When the value is ≥0.8 and the volume of the first fermentation broth is ≥75% of the volume of the first fermentation tank, 60% of the first fermentation broth is transferred to the second fermentation tank for second fermentation to obtain valine fermentation broth.

[0048] In the present invention, the height-to-diameter ratio of the second fermenter is (2-4):1. As an embodiment, the height-to-diameter ratio of the second fermenter of the present invention is 2.5:1. As an embodiment, the second fermenter of the present invention includes a tank wall, a temperature-controlled internal coil, a feed port, a feed port, a discharge port, an air inlet, an exhaust port, a stirring device, and a driving device. The feed port and the exhaust port are arranged at the top of the fermenter cavity, the temperature-controlled internal coil is distributed around the fermenter cavity, the feed port, the discharge port, and the air inlet are arranged at the bottom of the fermenter cavity, the driving device is arranged at the top of the fermenter and is connected to the stirring device, the stirring device extends from the top of the fermenter to the bottom of the tank cavity, and the stirring device is provided with a stirring paddle. The present invention has no strict requirements on the source of the second fermenter, and conventional purchase is sufficient.

[0049] In the present invention, during the second fermentation, the tail air from the first fermentor is used as an air source, and a fermentation feed is added to maintain a glucose concentration of 0.2-0.3 wt.% in the fermentation broth of the second fermentor. Using the tail air from the first fermentor as the air source for the valine fermentation process can conserve air, reduce the size of the air compressor, save electricity, and reduce the pressure on exhaust gas treatment. Compared to using external air, this can also reduce the risk of bacterial contamination.

[0050] In one embodiment, the temperature of the second fermentation culture of the present invention is 35°C to 38°C; in another embodiment, the temperature of the second fermentation culture of the present invention is 37°C. In one embodiment, the dissolved oxygen content of the fermentation broth of the second fermentation culture of the present invention is maintained below 5%; in another embodiment, the dissolved oxygen content of the fermentation broth of the second fermentation culture of the present invention is maintained below 2%. In one embodiment, the stirring frequency of the second fermentation culture of the present invention is 15 to 30 r / min; in another embodiment, the stirring frequency of the second fermentation culture of the present invention is 25 r / min. The second fermentation culture of the present invention is anaerobic fermentation.

[0051] In one embodiment, the fermentation feed of the present invention comprises the following components at the following concentrations: 3-5 g / L peptone, 300-350 g / L glucose, 1-2 g / L magnesium sulfate, 1.5-2 g / L potassium dihydrogen phosphate, 12-15 mg / L biotin, 20-25 mg / L cobalt chloride, and 5-8 mg / L copper sulfate. In another embodiment, the fermentation feed of the present invention comprises the following components at the following concentrations: 3 g / L peptone, 300 g / L glucose, 1.5 g / L magnesium sulfate, 1.5 g / L potassium dihydrogen phosphate, 15 mg / L biotin, and 20 mg / L cobalt chloride. In one embodiment, the solvent of the fermentation feed of the present invention is water.

[0052] In the present invention, after 60-65% of the first fermentation broth is transferred to the first fermentation tank, the nutrients described in the above technical solution are added to the first fermentation tank to continue fermentation and culture; during the continued fermentation and culture, the glucose concentration in the fermentation broth in the first fermentation tank is maintained at 0.5-0.8 wt.%, until the OD value of the fermentation broth in the first fermentation tank reaches 0.5-0.8 wt.%. 610nm The value is ≥ 0.8, and the volume of the fermentation liquid is ≥ 75% of the volume of the first fermentation tank. The composition of the fermentation medium and supplementary nutrients of the present invention has been described above and will not be repeated here.

[0053] In one embodiment, the temperature of the continued fermentation culture of the present invention is 36.3-36.7°C; in another embodiment, the temperature of the continued fermentation culture of the present invention is 36.5°C. In one embodiment, the stirring frequency of the continued fermentation culture of the present invention is 35-60 r / min; in another embodiment, the stirring frequency of the continued fermentation culture of the present invention is 50 r / min. In one embodiment, the dissolved oxygen content of the fermentation broth during the continued fermentation culture of the present invention is maintained at 15%-30%; in another embodiment, the dissolved oxygen content of the fermentation broth during the continued fermentation culture of the present invention is maintained at 20%. In one embodiment, the ventilation ratio is adjusted to maintain the dissolved oxygen content of the fermentation broth during the continued fermentation culture. In one embodiment, the ventilation ratio of the present invention is 0.25-1.0 L / (L·min); in another embodiment, the ventilation ratio of the present invention is 0.3-0.8 L / (L·min); in another embodiment, the ventilation ratio of the present invention is 0.5 L / (L·min).

[0054] After obtaining the valine fermentation broth, the present invention extracts the valine fermentation broth to obtain valine. As an embodiment, when the valine content in the valine fermentation broth is ≥0.8%, the present invention collects the valine fermentation broth. As an embodiment, the present invention collects the valine fermentation broth, repeats the steps of the first fermentation culture in the first fermentation tank, transferring the fermentation broth in the first fermentation tank to the second fermentation tank, supplementing the fermentation medium in the first fermentation tank to continue fermentation culture, the second fermentation culture in the second fermentation tank, and preparing and collecting the valine fermentation broth, until the fermentation efficiency of the second fermentation tank decreases or the first fermentation tank is contaminated with bacteria. As an embodiment, the number of repetitions of the present invention is 30 to 80 times; as another embodiment, the number of repetitions of the present invention is 40 to 70 times; as another embodiment, the number of repetitions of the present invention is 50 to 60 times.

[0055] As an embodiment, the extraction steps of the present invention include: performing a first filtration on the valine fermentation broth using a ceramic membrane to collect a first filtrate; performing a second filtration on the first filtrate using a nanofiltration membrane to collect a second filtrate; and sequentially concentrating, centrifuging and drying the second filtrate to obtain valine.

[0056] In one embodiment, the ceramic membrane of the present invention has a core diameter of 30 to 200 nm; in another embodiment, the ceramic membrane has a core diameter of 50 nm. In one embodiment, the ceramic membrane of the present invention has a transmembrane pressure differential of ≤10.0 bar; in another embodiment, the transmembrane pressure differential of the ceramic membrane is 6 bar.

[0057] In one embodiment, the nanofiltration membrane of the present invention has a core of 200-1000 DaI; in another embodiment, the nanofiltration membrane has a core of 300-800 DaI; in another embodiment, the nanofiltration membrane has a core of 400-600 DaI; and in another embodiment, the nanofiltration membrane has a core of 500 DaI. In one embodiment, the transmembrane pressure of the nanofiltration membrane of the present invention is 18-20 bar; in another embodiment, the transmembrane pressure of the nanofiltration membrane of the present invention is 19 bar.

[0058] In one embodiment, the concentration described herein is evaporation concentration, and the output density of the evaporation concentration is 1.15 to 1.22 g / mL. In one embodiment, the evaporation concentration described herein utilizes a multi-effect rising film evaporator and / or a multi-effect falling film evaporator; in another embodiment, the evaporation concentration described herein utilizes a multi-effect falling film evaporator.

[0059] In one embodiment, the centrifuge speed of the present invention is 800-1500 rpm; in another embodiment, the centrifuge speed of the present invention is 1250 rpm. In one embodiment, the separation factor of the centrifuge of the present invention is 631-1007; in another embodiment, the separation factor of the centrifuge of the present invention is 698. In one embodiment, the centrifugation time of the present invention is 10-15 minutes; in another embodiment, the centrifugation time of the present invention is 12 minutes. In one embodiment, the centrifugation of the present invention uses a flat-plate centrifuge with a centrifuge drum diameter of 1000-1600 mm.

[0060] In one embodiment, the drying time of the present invention is 1 to 15 minutes; in another embodiment, the drying time of the present invention is 5 minutes. In one embodiment, the drying temperature of the present invention is 120 to 150°C; in another embodiment, the drying temperature of the present invention is 146°C. In one embodiment, the drying of the present invention is performed using an airflow drying tower.

[0061] The present invention also provides a fermentation device for producing valine, comprising a first fermentation tank and a second fermentation tank; an exhaust port of the first fermentation tank is connected to an air inlet of the second fermentation tank through a pipeline;

[0062] The discharge port of the first fermentation tank is connected to the feed port of the second fermentation tank through a pipeline;

[0063] The height-to-diameter ratio of the first fermentation tank is (4-6):1; the height-to-diameter ratio of the second fermentation tank is (2-4):1.

[0064] To further illustrate the present invention, a method for producing valine by fermentation and a fermentation device for producing valine provided by the present invention are described in detail below with reference to the following examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0065] Example 1

[0066] A method for producing valine by fermentation comprises the following steps:

[0067] 1. Culture medium preparation

[0068] Fermentation medium: water as solvent, composed of yeast powder 2.5g / L, peptone 1.5g / L, corn steep liquor 5g / L, betaine 1.2g / L, glucose 12g / L, magnesium sulfate 0.6g / L, potassium dihydrogen phosphate 2g / L, biotin 3.8mg / L and calcium pantothenate 20mg / L, sterilized at 121℃ for 30min.

[0069] Nutrient medium: water as solvent, composed of yeast powder 0.5g / L, peptone 13g / L, corn steep liquor 4g / L, betaine 3g / L, glucose 75g / L, magnesium sulfate 0.5g / L and potassium dihydrogen phosphate 0.8g / L, sterilized at 130℃ for 7min;

[0070] Preparation of fermentation material: using water as solvent, consisting of 3 g / L peptone, 300 g / L glucose, 1.5 g / L magnesium sulfate, 1.5 g / L potassium dihydrogen phosphate, 15 mg / L biotin, and 20 mg / L cobalt chloride, sterilized at 130°C for 10 min.

[0071] 2. Preparation of E. coli Seed Solution

[0072] Escherichia coli was inoculated into seed culture medium (yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L, glucose 100g / L) for seed culture until the bacterial OD 610nm When the value is 10-12, Escherichia coli seed solution is obtained.

[0073] 3. Fermentation production of valine

[0074] (1) The fermentation tank with a height-to-diameter ratio of 6:1 was used as the first fermentation tank, and the fermentation tank with a height-to-diameter ratio of 2.5:1 was used as the second fermentation tank. The first and second fermentation tanks were cleaned and sterilized separately. Fermentation medium of 15% of the volume of the first fermentation tank was added to the first fermentation tank and sterilized, and 0.02% Escherichia coli seed liquid was added to start fermentation culture. The fermentation process maintained a temperature of 36.5±0.2℃, a stirring frequency of 50r / min, and the ventilation ratio was adjusted to keep the dissolved oxygen content of the fermentation liquid at 15%~30%. When the initial sugar was exhausted, nutrients were added to make the glucose concentration in the fermentation liquid 0.5~0.8% (w / v), and the OD of the fermentation liquid in the first fermentation tank was 0.5~0.8%. 610nm The value increases to above 0.8.

[0075] (2) When the volume of the fermentation liquid in the first fermentation tank reaches 75% of the volume of the first fermentation tank, 60% of the fermentation liquid is transferred to the second fermentation tank, and the nutrients described in step (1) are continuously added to the first fermentation tank to continue the fermentation culture; during the process of continuing the fermentation culture, the glucose concentration in the fermentation liquid in the first fermentation tank is maintained at 0.5-0.8 wt.%, until the OD value of the fermentation liquid in the first fermentation tank reaches 0.5-0.8 wt.%. 610 The nm value is higher than 0.8, and the volume of the fermentation liquid in the first fermentation tank reaches 75% of the volume of the first fermentation tank; the fermentation process is continued to maintain a temperature of 36.5±0.2°C, a stirring frequency of 50r / min, and a ventilation ratio is adjusted to keep the dissolved oxygen content of the fermentation liquid at 15%~30%.

[0076] (3) The second fermentation tank uses the tail wind of the first fermentation tank as the air source for the fermentation process, and continuously adds fermentation materials to make the glucose concentration in the fermentation liquid in the second fermentation tank 0.2~0.3% (w / v) for valine fermentation. When the valine content in the fermentation liquid reaches more than 8.0%, the material is discharged to the ceramic membrane filtration process, and the second fermentation tank is cleaned and sterilized again.

[0077] (4) Repeat the process of feeding the first fermentation tank, discharging the material to the second fermentation tank, receiving the material in the second fermentation tank, feeding the small material to ferment and produce valine, and discharging the material to the subsequent extraction process.

[0078] (5) When the fermentation efficiency of the second fermentation tank decreases or the first fermentation tank is contaminated with bacteria, the first fermentation tank and the second fermentation tank are cleaned and sterilized again, and the subsequent process is carried out to achieve semi-continuous fermentation of valine.

[0079] (6) The fermentation liquid discharged from the second fermentation tank is filtered through a 50 nm ceramic membrane and a 200 Da1 nanofiltration membrane, concentrated by a multi-effect falling film evaporator, separated by a flat scraper bottom discharge centrifuge, dried in an airflow drying tower and packaged to obtain the valine product.

[0080] One fermentation cycle was defined as the fermentation starting from step (1) in the first fermenter to the end of the fermentation in the second fermenter, where the material was discharged to the ceramic membrane filtration process. Fermentation was terminated when the efficiency of valine production in the second fermenter decreased. In this embodiment, a total of 73 fermentation cycles were performed, with an average fermentation cycle of 6.52 h for valine production and an average valine conversion rate of 52.47%. The specific fermentation cycles are shown in Table 1.

[0081] Example 2

[0082] A method for producing valine by fermentation is similar to that of Example 1, except that a fermentor with a height-to-diameter ratio of 4:1 is used as the first fermentor, and a fermentor with a height-to-diameter ratio of 2.5:1 is used as the second fermentor. The first and second fermentors are cleaned and sterilized separately. Fermentation medium, equivalent to 10% of the volume of the first fermentor, is added to the first fermentor and sterilized. A 0.02% Escherichia coli seed solution is then added to the first fermentor and fermentation is carried out as in Example 1.

[0083] In this example, a total of 69 fermentation cycles were carried out. The average fermentation cycle for producing valine was 6.87 h, and the average valine conversion rate reached 50.99%. The specific fermentation cycles are shown in Table 1.

[0084] Comparative Example 1

[0085] (1) Add E. coli seeds to 1m 3 The first-level seed tank is used for cultivation.

[0086] (2) Waiting for OD 610nm When the value reaches 0.4 or above, transplant to 50m 3 The secondary seed tank is used for expansion culture.

[0087] (3) Wait for the secondary seed to OD 610nm When the value reaches 0.8 or above, transplant to a height-to-diameter ratio of 3.5:1 and a total volume of 340m 3 The fermentation tank was used for fermentation to produce valine. During the fermentation process, the temperature was maintained at 36.5±0.1℃, the stirring frequency was 45r / min, the ventilation ratio was adjusted, and the dissolved oxygen content of the fermentation liquid was kept above 25% in the early stage of fermentation. 610 When the value reaches 0.4, reduce the stirring and ventilation volume to reduce the dissolved oxygen to below 2% and continue the fermentation culture.

[0088] (4) When the valine content in the fermentation tank reaches 8.0% or above, the fermentation is stopped, and the fermentation liquid is filtered through a 50nm ceramic membrane and a 200Dal nanofiltration membrane, concentrated by a multi-effect falling film evaporator, separated by a flat scraper bottom discharge centrifuge, dried in an airflow drying tower, and packaged to obtain a qualified valine product.

[0089] In this comparative example, the fermentation period of valine was 23 h, and the conversion rate was 51.85%.

[0090] Comparative Example 2

[0091] A method for producing valine by fermentation was similar to that described in Example 1, except that a fermentor with a height-to-diameter ratio of 2.5:1 was used as the first fermentor, and a fermentor with a height-to-diameter ratio of 6:1 was used as the second fermentor. The first and second fermentors were cleaned and sterilized separately. Fermentation medium, equivalent to 10% of the volume of the first fermentor, was added to the first fermentor and sterilized. A 0.02% Escherichia coli seed solution was then added to the first fermentor and fermentation was carried out as described in Example 1.

[0092] Because the first fermentation tank has a relatively small height-to-diameter ratio, the proportion of oxygen in the air dissolved in the fermentation liquid after entering the fermentation tank is relatively small, resulting in a ventilation volume of about 40% higher than that of Example 1 in this comparative example. The second fermentation tank has a large height-to-diameter ratio, oxygen is completely dissolved, and this part of the fermentation requires a lower dissolved oxygen value. Therefore, only about 50% of the air discharged from the first fermentation tank is passed into the second fermentation tank, and the rest is discharged into the air after treatment, resulting in waste.

[0093] This comparative example carried out a total of 75 fermentation cycles, with an average fermentation cycle for producing valine of 9.51 h and an average valine conversion rate of 50.99%. The specific fermentation cycles are shown in Table 1.

[0094] Table 1 Time and conversion rate of different fermentation cycles of Examples 1-2 and Comparative Example 2

[0095]

[0096]

[0097] It can be seen from the above content that the technical solution provided by the present invention has high valine fermentation efficiency, short production chain, short production cycle and low cost.

[0098] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for producing valine by fermentation, characterized in that: The steps include: (1) adding 10% to 15% of the volume of the first fermentation tank to the first fermentation tank, inoculating the seed liquid to perform the first fermentation culture, and obtaining the first fermentation liquid; The height-to-diameter ratio of the first fermentation tank is (4-6):1; The seed liquid is the seed liquid of a valine-producing strain; During the first fermentation culture, after the initial sugar in the fermentation medium is exhausted, nutrients are added to make the glucose concentration in the fermentation broth 0.5-0.8 wt.%; (2) When the OD of the first fermentation liquid in step (1) 610nm When the value is ≥0.8 and the volume of the first fermentation broth is ≥75% of the volume of the first fermentation tank, 60%-65% of the first fermentation broth is transferred to a second fermentation tank for a second fermentation culture to obtain a valine fermentation broth; the height-to-diameter ratio of the second fermentation tank is 2.5:1; during the second fermentation culture, the tail wind of the first fermentation tank is used as an air source, and fermentation feed is fed to ensure that the glucose concentration of the fermentation broth in the second fermentation tank is 0.2-0.3 wt.%; After 60% to 65% of the first fermentation liquid is transferred to the first fermentation tank, the nutrients in step (1) are added to the first fermentation tank to continue fermentation and cultivation; during the continued fermentation and cultivation, the glucose concentration in the fermentation liquid in the first fermentation tank is maintained at 0.5 to 0.8 wt.%, until the OD value of the fermentation liquid in the first fermentation tank reaches 0.5 to 0.8 wt.%. 610nm The value is ≥0.8, and the volume of the fermentation liquid is ≥75% of the volume of the first fermentation tank; (3) Extracting the valine fermentation broth described in step (2) to obtain valine.

2. The method according to claim 1, characterized in that The temperature of the first fermentation culture is 35-37°C, the stirring frequency is 35-60 r / min, and the dissolved oxygen content of the fermentation liquid is maintained at 15%-30%; The temperature of the continued fermentation culture is 35-37°C, the stirring frequency is 35-60 r / min, and the dissolved oxygen content of the fermentation liquid is maintained at 15%-30%; The temperature of the second fermentation culture is 35-38° C., the stirring frequency is 15-30 r / min, and the dissolved oxygen content of the fermentation liquid is maintained below 5%.

3. The method according to claim 1, characterized in that The fermentation medium of step (1) and step (2) respectively comprises the following components in the following concentrations: yeast powder 1-3 g / L, peptone 1-2 g / L, corn steep liquor 5-7 g / L, betaine 1-1.2 g / L, glucose 10-15 g / L, magnesium sulfate 0.5-0.8 g / L, potassium dihydrogen phosphate 1.5-2 g / L, biotin 3-6 mg / L and calcium pantothenate 20-25 mg / L.

4. The method according to claim 1, wherein The nutrients in step (1) and step (2) respectively include the following components in concentrations: yeast powder 0.5-1 g / L, peptone 1-1.5 g / L, corn steep liquor 3-5 g / L, betaine 2-3 g / L, glucose 70-75 g / L, magnesium sulfate 0.5-0.8 g / L and potassium dihydrogen phosphate 0.8-1 g / L.

5. The method according to claim 1, wherein The fermentation material in step (2) includes the following components in concentrations: peptone 3-5 g / L, glucose 300-350 g / L, magnesium sulfate 1-2 g / L, potassium dihydrogen phosphate 1.5-2 g / L, biotin 12-15 mg / L, cobalt chloride 20-25 mg / L, and copper sulfate 5-8 mg / L.

6. The method according to claim 1, characterized in that The extraction step comprises: performing a first filtration on the valine fermentation broth using a ceramic membrane to collect a first filtrate; performing a second filtration on the first filtrate using a nanofiltration membrane to collect the second filtrate; sequentially concentrating, centrifuging and drying the second filtrate to obtain valine; The ceramic membrane has a core thickness of 30-200 nm and a transmembrane pressure difference of ≤10.0 bar; The membrane core of the nanofiltration membrane is 200~1000Dal, and the transmembrane pressure difference is 18~20bar.

7. The method according to claim 6, characterized in that The concentration is evaporation concentration, and the output density of the evaporation concentration is 1.15-1.22 g / mL.

8. The method according to any one of claims 1 to 7, characterized in that The method further comprises: after obtaining the valine fermentation broth, collecting the valine fermentation broth, and repeating step (2) until the fermentation efficiency of the second fermentation tank decreases or the first fermentation tank is contaminated with bacteria.

9. The method according to claim 8, characterized in that The number of repetitions is 30 to 80 times.

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