CN112481321B - Process for producing granular threonine - Google Patents
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Abstract
The invention belongs to the technical field of amino acid production, and relates to a production process of granular threonine, which comprises the following steps: step 1) fermentation, step 2) centrifugation, step 3) membrane filtration, step 4) concentration crystallization, step 5) separation of crystals, step 6) drying, step 7) primary mother liquor crystallization and separation, step 8) chromatographic separation, step 9) crystallization and separation, and step 10) granulation.
Description
Technical Field
The invention belongs to the technical field of amino acid production, and particularly relates to a production process of granular threonine.
Background
Threonine (abbreviated as Thr) belongs to the technical name of 2-amino-3-hydroxybutyric acid, which belongs to aliphatic amino acid and is slightly sweet, and has four stereoisomers because of the fact that Threonine has a similar structure to threose and contains two asymmetric chiral carbon atoms in the molecule. Of these, only L-threonine has biological activity. L-threonine is one of eight essential amino acids, with a relative molecular mass of 119.12, melting point 253-257 ℃. L-threonine is easily soluble in water, insoluble in chloroform, ethanol, diethyl ether, etc.
L-threonine plays an important role in promoting the growth and development of human bodies and maintaining normal physiological functions, so that the L-threonine is widely applied to the fields of foods, medicines and the like. Nutritional additives play an important role in the food industry because of the large throughput and wide distribution area of current foods, the nutritional ingredients in foods are susceptible to damage and partial loss. Wherein threonine is used as an amino acid additive, which can effectively enrich nutrition components and improve oxidation resistance of food. Meanwhile, threonine and glucose co-heat also play a good role in flavoring. Secondly, threonine is also an essential amino acid for livestock and poultry. And methionine, lysine and tryptophan and are known as tetraaa feed additives. Threonine is added into livestock and poultry feed, so that the amino acid types in the feed can be effectively enriched, the nitrogen utilization rate is improved, the synthesis of protein is promoted, and the production cost of the feed is reduced. Research shows that the threonine can effectively improve and maintain the feed intake and the immunity of livestock and poultry and regulate the fat metabolism of animals. In addition, amino acids are also in great demand in the medical field. Threonine is an important precursor for synthesizing various biological components in a human body and has the capability of improving immunity, so that threonine is used as one of components for large transfusion of amino acid and is widely used for adjuvant therapy of various diseases before and after operation. In addition, threonine iron salt is also a widely used anti-anemia agent.
Currently, threonine production methods mainly include three methods, namely fermentation, protein hydrolysis and chemical synthesis, and microbial fermentation has become a mainstream method for threonine production. The main production strains of L-threonine include Escherichia coli, corynebacterium glutamicum, serratia marcescens, etc. In addition, since E.coli has the advantages of easy cultivation, short fermentation time, clear genetic background, etc., E.coli has been one of the most dominant strains for L-threonine production in the fermentation industry so far.
The applicant has made extensive studies on threonine fermentation. The literature is exemplified as follows:
"CN110904167A, L-threonine fermentation process optimization method, 2020.3.24" discloses an L-threonine fermentation process optimization method comprising the steps of: inoculating seed solution of L-threonine producing strain into fermentation tank containing fermentation medium according to 1-2% inoculum size, fermenting at 35-37deg.C under stirring at 300-500rpm, controlling dissolved oxygen content to 15-20% by aeration and stirring, defoaming with foam enemy, fermenting for 36 hr, stopping fermentation, and collecting fermentation liquid. The invention improves the yield of L-threonine by optimizing the fermentation method. "CN110894522A, a corn husk hydrolysate and its use in the fermentative preparation of threonine, 2020.3.20" discloses a corn husk hydrolysate prepared according to the following process: step 1) corn husk pretreatment, step 2) ultrasonic-assisted hydrochloric acid hydrolysis, step 3) microbial hydrolysis, step 4) hydrolysis of thalli, step 5) concentration and steam treatment. The hydrolysate contains polypeptide, amino acid and reducing sugar, can be applied to threonine fermentation, reduces cost and improves fermentation efficiency.
"CN110846348A, a preparation method of threonine fermentation medium, 2020.02.28" discloses a preparation method of threonine fermentation medium, comprising the steps of: glucose, corn steep liquor, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, N-methyl aspartic acid, methionine, ferrous sulfate heptahydrate and manganese sulfate monohydrate are sequentially added into purified water, stirred and dissolved, and the pH value is regulated to 6.5-7.0. According to the invention, the fermentation medium is optimized, and the N-methyl aspartic acid and methionine are added in the fermentation process, so that the fermentation efficiency of threonine is improved, the fermentation period is shortened, and the cost is saved.
"CN110551773A, a method for replacing yeast powder with soybean meal enzymatic hydrolysate in threonine production, 2019.12.10", comprises the following steps: 1) Preparing soybean meal enzymolysis liquid, and 2) fermenting the strain. Compared with yeast extract, the invention has the advantages that the threonine yield and dry matter in fermentation liquor are improved, and the cost is relatively low.
"CN110396530A, a method for improving threonine yield and 2019.11.1", discloses a method for improving threonine yield and yield, comprising the steps of: step 1) preparation of fermentation medium, step 2) threonine fermentation, step 3) separation, crystallization and drying. The method reduces the cost and improves the yield and the yield.
"CN110004192A, a method for producing granular threonine, 2019.7.12" discloses a method for producing granular threonine, comprising the steps of: step 1) fermentation, step 2) centrifugation, and step 3) preparation of granular threonine. The method can improve the threonine content of the particles, has simple and feasible process and wide application prospect.
"CN109609566A, a method for improving threonine production, 2019.04.12" discloses a method for improving threonine production, comprising the steps of: inoculating threonine producing escherichia coli engineering bacteria seed liquid into a fermentation tank containing a fermentation medium according to an inoculum size of 10-12% for fermentation, wherein the temperature is 30-34 ℃, the tank pressure is 0.03-0.04MPa, the ventilation amount is 0.4-0.6vvm, the rotation speed is 100rpm, the fermentation time is 30-36h, then adding inositol 100-200mg/L, continuing fermentation culture for 24-30h, stopping fermentation, and collecting fermentation liquor. The invention improves threonine output through optimizing fermentation technology.
Other relevant research institutions have also studied, for example:
"Jilin university CN107760734A, a method for improving threonine yield and application thereof, 2018.03.06" discloses a method for improving threonine yield, wherein the method for improving threonine yield is to add a mixed fermentation promoter consisting of betaine hydrochloride, VB4 and VB3 in the L-threonine fermentation process, so that the improvement of L-threonine yield by 10-12% can be realized.
"Tianjin university of science and technology CN107012181A, a threonine fermentation medium and threonine clean production process, 2017.08.04" discloses a threonine fermentation medium and threonine clean production process, the fermentation medium does not contain corn steep liquor and yeast powder, various amino acids, nucleosides and trace elements are added to achieve substitution effect, and the medium is utilized to ferment threonine clear liquid so as to improve the sugar acid conversion rate and extraction yield.
By analysis, the conventional threonine production process mainly has the following defects: the fermentation yield is low, and the fermentation process is not comprehensively and systematically optimized; the traditional powdery threonine is easy to absorb moisture and agglomerate due to smaller granularity, is unfavorable for transportation and storage, has poor fluidity and large dust in the feed adding and using process, is unfavorable for adding, and brings certain limit to the development of threonine industry. There is a need to develop a novel process for the production of granular threonine to compensate for the above-mentioned disadvantages.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a production process of granular threonine.
In order to achieve the above object, the present invention is achieved by the following technical scheme:
a process for the production of granular threonine comprising the steps of: step 1) fermentation, step 2) centrifugation, step 3) membrane filtration, step 4) concentration crystallization, step 5) separation of crystals, step 6) drying, step 7) primary mother liquor crystallization and separation, step 8) chromatographic separation, step 9) crystallization and separation, and step 10) granulation.
In particular, the method comprises the steps of,
the production process comprises the following steps:
step 1) fermentation: inoculating threonine-producing escherichia coli engineering bacteria seed liquid into a fermentation tank containing a fermentation medium according to an inoculum size of 6%, fermenting at a temperature of 30 ℃, a tank pressure of 0.04MPa, a ventilation amount of 0.6vvm, a rotating speed of 100rpm, a fermentation time of 72 hours, stopping fermentation, and collecting fermentation liquor;
step 2) centrifuging: centrifuging the fermentation liquor at 4500-5000rpm for 4-5min by a disc centrifuge, and collecting supernatant liquid and precipitate;
step 3) membrane filtration: performing membrane filtration on the upper liquid obtained in the step 2) by utilizing a ceramic membrane, and collecting ceramic membrane concentrated solution and ceramic membrane permeate; the obtained ceramic membrane concentrated solution enters a horizontal decanter centrifuge for treatment, the rotating speed is 2000r/min, and the inlet flow is 16m 3 And (h) combining the supernatant obtained after centrifugation with the ceramic membrane permeate to obtain a combined permeate;
step 4) concentrating and crystallizing: the combined permeate liquid obtained in the step 3) enters an evaporator, is evaporated and concentrated to one fourth of the volume of the stock solution, and then enters a crystallization tank for cooling and crystallization;
step 5) isolating crystals: centrifuging with a centrifuge, increasing the rotational speed of the centrifuge from 400-500r/min to 900-1000r/min at a speed of 10r/s, maintaining the rotational speed of 900-1000r/min, centrifuging for 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystals to obtain a powdery threonine product;
step 7) primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, centrifuging by a horizontal decanter centrifuge, and collecting crystals and secondary mother liquor;
step 8) chromatographic separation: performing chromatographic separation on the secondary mother liquor by using a sequential simulated moving bed to remove sugar, salt and pigment, thereby obtaining a threonine-rich separating liquid;
step 9) crystallization and separation: crystallizing the separating liquid obtained in the step 8), centrifugally separating crystals, then combining the crystals with the crystals obtained in the step 7), and pumping the crystals into a crystallization tank in the step 4) for crystallization;
step 10) granulating: compressing the powdery threonine obtained in the step 6) into thin strips by using a rolling granulator, gradually reducing the pressure born by the material after extrusion forming, falling off downwards under the action of gravity, and entering a crushing and granulating system; then the mixture enters a return auger according to the granularity requirement, passes through a sieve with 10 meshes and a sieve with 30 meshes in sequence, screens out coarse and fine particles, collects the particle products with the particle size of between 10 meshes and 30 meshes, and packages the products to obtain the finished product; the undersize powder is returned again to the granulation.
Further, the components of the fermentation medium are: 50g/L of glucose, 20g/L of glycerol, 20g/L of corn steep liquor, 2g/L of ammonium sulfate, 0.2g/L of monopotassium phosphate, 0.2g/L of dipotassium phosphate, 100mg/L of magnesium sulfate heptahydrate, 60-80mg/L of 4-hydroxy-3-methoxybenzoic acid, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate and pH value of 6.5.
Further, the method comprises the steps of,
the fermentation in the step 1) further comprises the following steps:
1) Monitoring the concentration of glucose in the fermentation liquid, and controlling the sugar content to be 2% by feeding the first nutrient solution until the fermentation is finished;
2) Monitoring the pH of the fermentation liquid, and controlling the pH to be 6.5 by adding 20% ammonia water until the fermentation is finished;
3) And (3) after fermentation for about 24 hours, feeding a second nutrient solution into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquid until the fermentation is finished.
Further, the method comprises the steps of,
the conditions of the membrane filtration are as follows: membrane module dialysate flow 19m 3 And/h, the temperature of the membrane equipment is not higher than 80 ℃, the inlet pressure of the membrane is 0.3MPa, the outlet pressure of the membrane is 0.10MPa, the top pressure of the equipment is 0.15MPa, and the osmotic side pressure (high pressure side) is as follows: 0.15MPa, the permeate side pressure (low pressure side) is: 0.1MPa, the clear liquid flow (high pressure side) is: 15m 3 Flow rate of clear liquid (low pressure side): 10m 3 /h。
Further, the roller pressure of the roller granulator is controlled to be 175-200 kg, and the roller spacing is controlled to be 2.2-2.5mm.
Further, the method comprises the steps of,
the first nutrient solution comprises the following components: glucose 500g/L, methionine 1-5g/L, isoleucine 1-5g/L.
Further, the method comprises the steps of,
the second nutrient solution comprises the following components: 5-20g/L of sodium acetate and 1-5g/L of adenosine.
Still further, the method further comprises the steps of,
the first nutrient solution comprises the following components: glucose 500g/L, methionine 2g/L, isoleucine 2g/L.
Still further, the method further comprises the steps of,
the second nutrient solution comprises the following components: 20g/L of sodium acetate and 5g/L of adenosine.
The invention has the following advantages, including but not limited to:
the pentose phosphate pathway provides glyceraldehyde triphosphate to enter a threonine synthesis pathway, and can also provide a large amount of NADPH for threonine synthesis, so that threonine synthesis is promoted; according to the invention, by adding 4-hydroxy-3-methoxybenzoic acid into the fermentation medium, various key enzymes in the pentose phosphate pathway including glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase can be activated, so that the pentose phosphate pathway is enhanced, and further, the sugar acid conversion rate and the fermentation efficiency of threonine are improved.
The methionine and lysine synthetic pathways can split threonine synthesis, and with the increase of threonine yield, the downstream product isoleucine begins to accumulate, so that loss of carbon flux is caused, and the reduction of metabolic flux entering the methionine and lysine pathways can promote the increase of threonine metabolic flux; the invention utilizes the K12dapA escherichia coli to belong to a strain producing lysine deficiency, and the regulation and control of lysine paths are not needed; when the glucose consumption is less than 2%, the strain proliferation is slowed down, and at the moment, a large amount of metabolites are synthesized, and glucose is supplemented by glucose, methionine and isoleucine for metabolism of the glucose-supplementing sugar co-strain, wherein the methionine and the isoleucine can have a certain feedback inhibition effect on the methionine and the isoleucine, and the normal metabolism and proliferation of the strain cannot be influenced.
Metabolites such as acetic acid and the like can be produced in the middle and late growth period of the cells, so that not only is the carbon flux lost, but also the cells are damaged, and the cell activity is affected, the acetic acid synthesis pathway can be inhibited by feeding sodium acetate, the production of acetic acid is reduced, and the amount of converted ATP can be increased by providing a certain amount of adenosine, so that the strain aging too fast caused by insufficient intracellular ATP supply is avoided.
The invention adjusts a plurality of factors and metabolic points in threonine synthesis paths, and aims to optimize the fermentation process to the greatest extent and improve the fermentation efficiency and the sugar acid conversion rate.
In the invention, in the preparation of the powdery threonine into the granular threonine, a disc centrifuge is adopted for centrifugal treatment, so that the pressure of membrane separation is reduced; the invention prepares threonine into granular threonine, which can improve fluidity, facilitate preservation and transportation, control solubility, improve quality and added value of threonine and increase economic value.
Drawings
Fig. 1: influence of 4-hydroxy-3-methoxybenzoic acid concentration on threonine content in fermentation broth;
fig. 2: influence of 4-hydroxy-3-methoxybenzoic acid concentration on sugar acid conversion;
fig. 3: influence of the first nutrient solution component on the threonine content of the fermentation broth;
fig. 4: effect of the second nutrient solution component on threonine content in the fermentation broth.
Detailed Description
Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced with modification and alteration and combination of the products and methods described herein without departing from the spirit and scope of the invention. The present invention will be described in detail with reference to examples.
Example 1
A process for the production of granular threonine comprising the steps of:
step 1) fermentation: inoculating Escherichia coli engineering bacteria K12DeltadapA seed solution (prepared by inoculating Escherichia coli engineering bacteria into seed culture medium according to 10% inoculum size, culturing at 30deg.C and shaking table rotation speed of 180r/min until the concentration of seed solution is 1×10) 9 cfu/mL, the seed culture medium comprises the following components: 5g of yeast extract, 20g of glucose, 0.5g of ammonium sulfate, 0.1g of potassium dihydrogen sulfate, 0.01g of ferrous sulfate heptahydrate, 0.02g of magnesium sulfate heptahydrate, the balance of water and pH value of 6.5-7.0), inoculating the yeast extract into a fermentation tank containing a fermentation medium according to an inoculation amount of 6% to ferment, wherein the temperature is 30 ℃, the tank pressure is 0.04MPa, the ventilation amount is 0.6vvm, the rotating speed is 100rpm, the fermentation time is 72 hours, stopping fermentation, and collecting fermentation liquor;
the components of the fermentation medium are as follows: glucose 50g/L, glycerin 20g/L, corn steep liquor 20g/L, ammonium sulfate 2g/L, monopotassium phosphate 0.2g/L, dipotassium phosphate 0.2g/L, magnesium sulfate heptahydrate 100mg/L, 4-hydroxy-3-methoxybenzoic acid 80mg/L, ferrous sulfate heptahydrate 10mg/L, manganese sulfate monohydrate 10mg/L and pH value 6.5; during the fermentation process:
1) Monitoring the concentration of glucose in the fermentation liquid, and controlling the sugar content to be 2% by feeding a first nutrient solution until the fermentation is finished, wherein the first nutrient solution comprises the following components: glucose 500g/L, methionine 2g/L, isoleucine 2g/L;
2) Monitoring the pH of the fermentation liquid, and controlling the pH to be 6.5 by adding 20% ammonia water until the fermentation is finished;
3) And (3) after fermentation for about 24 hours, feeding a second nutrient solution into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquid until the fermentation is finished, wherein the second nutrient solution comprises the following components: 20g/L of sodium acetate and 5g/L of adenosine; step 2) centrifuging: centrifuging the fermentation liquor at 4500rpm for 5min by a disc centrifuge, and collecting supernatant liquid and precipitate;
step 3) membrane filtration: treating the upper liquid obtained in the step 2) by using a ceramic membrane, wherein the dialysate flow rate of a membrane assembly is 19m 3 And/h, the temperature of the membrane equipment is not higher than 80 ℃, the inlet pressure of the membrane is 0.3MPa, the outlet pressure of the membrane is 0.10MPa, the top pressure of the equipment is 0.15MPa, and the osmotic side pressure (high pressure side) is as follows: 0.15MPa, the permeate side pressure (low pressure side) is: 0.1MPa, the clear liquid flow (high pressure side) is: 15m 3 Flow rate of clear liquid (low pressure side): 10m 3 /h; the obtained ceramic membrane concentrated solution enters a horizontal decanter centrifuge for treatment, the rotating speed is 2000r/min, and the inlet flow is 16m 3 And (h) combining the supernatant obtained after centrifugation with a ceramic membrane permeate;
step 4) concentrating and crystallizing: the combined liquid obtained in the step 3) enters an evaporator, is evaporated and concentrated to one fourth of the volume of the stock solution, and then enters a crystallization tank for cooling and crystallization;
step 5) isolating crystals: centrifuging by using a centrifugal machine, increasing the rotation speed of the centrifugal machine from 400r/min to 900r/min at the speed of 10r/s, maintaining the rotation speed of 900r/min, centrifuging for about 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystals to obtain a powdery threonine product;
step 7) primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, centrifuging by a horizontal decanter centrifuge, and collecting crystals and secondary mother liquor;
step 8) chromatographic separation: performing chromatographic separation on the secondary mother liquor by using a sequential simulated moving bed, and removing impurities such as sugar, salt, pigment and the like to obtain a threonine-rich separation liquid;
step 9) crystallization and separation: crystallizing the separating liquid obtained in the step 8), centrifugally separating crystals, then combining the crystals with the crystals obtained in the step 7), and pumping the crystals into a crystallization tank in the step 4) for crystallization;
step 10) granulating: the powder threonine obtained in the step 6) is compressed into thin strips by a rolling granulator through adjusting the roller pressure and the roller spacing (the roller spacing is 2.2mm and the pressure is 175 kg), the pressure born by the material after extrusion forming is gradually reduced, and the material falls off downwards under the action of gravity and enters a crushing and granulating system; then the mixture enters a return auger according to the granularity requirement, passes through a sieve with 10 meshes and a sieve with 30 meshes in sequence, screens out coarse and fine particles, collects the particle products with the particle size of between 10 meshes and 30 meshes, and packages the products to obtain the finished product; the undersize powder is returned again to the granulation.
Example 2
A process for the production of granular threonine comprising the steps of:
step 1) fermentation: inoculating Escherichia coli engineering bacteria K12DeltadapA seed solution (inoculating Escherichia coli engineering bacteria into seed culture medium according to 10% inoculum size, culturing at 30deg.C and shaking table rotation speed of 180r/min until the concentration of seed solution is 1×10) 9 cfu/mL, the seed culture medium comprises the following components: 5g of yeast extract, 20g of glucose, 0.5g of ammonium sulfate, 0.1g of potassium dihydrogen sulfate, 0.01g of ferrous sulfate heptahydrate, 0.02g of magnesium sulfate heptahydrate, the balance of water and pH value of 6.5-7.0), inoculating the yeast extract into a fermentation tank containing a fermentation medium according to an inoculation amount of 6% to ferment, wherein the temperature is 30 ℃, the tank pressure is 0.04MPa, the ventilation amount is 0.6vvm, the rotating speed is 100rpm, the fermentation time is 72h, and the fermentation is stopped to collect fermentation liquor;
the components of the fermentation medium are as follows: glucose 50g/L, glycerin 20g/L, corn steep liquor 20g/L, ammonium sulfate 2g/L, monopotassium phosphate 0.2g/L, dipotassium phosphate 0.2g/L, magnesium sulfate heptahydrate 100mg/L, 4-hydroxy-3-methoxybenzoic acid 60mg/L, ferrous sulfate heptahydrate 10mg/L, manganese sulfate monohydrate 10mg/L and pH value 6.5; during the fermentation process:
1) Monitoring the concentration of glucose in the fermentation liquid, and controlling the sugar content to be 2% by feeding a first nutrient solution until the fermentation is finished, wherein the first nutrient solution comprises the following components: glucose 500g/L, methionine 3g/L, isoleucine 3g/L;
2) Monitoring the pH of the fermentation liquid, and controlling the pH to be 6.5 by adding 20% ammonia water until the fermentation is finished;
3) And (3) after fermentation for about 24 hours, feeding a second nutrient solution into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquid until the fermentation is finished, wherein the second nutrient solution comprises the following components: 15g/L of sodium acetate and 4g/L of adenosine; step 2) centrifuging: centrifuging the fermentation liquor at 4500rpm for 5min by a disc centrifuge, and collecting supernatant liquid and precipitate;
step 3) membrane filtration: treating the upper liquid obtained in the step 2) by using a ceramic membrane, wherein the dialysate flow rate of a membrane assembly is 19m 3 And/h, the temperature of the membrane equipment is not higher than 80 ℃, the inlet pressure of the membrane is 0.3MPa, the outlet pressure of the membrane is 0.10MPa, the top pressure of the equipment is 0.15MPa, and the osmotic side pressure (high pressure side) is as follows: 0.15MPa, the permeate side pressure (low pressure side) is: 0.1MPa, the clear liquid flow (high pressure side) is: 15m 3 Flow rate of clear liquid (low pressure side): 10m 3 /h; the obtained ceramic membrane concentrated solution enters a horizontal decanter centrifuge for treatment, the rotating speed is 2000r/min, and the inlet flow is 16m 3 And (h) combining the supernatant obtained after centrifugation with a ceramic membrane permeate;
step 4) concentrating and crystallizing: then the mixture enters an evaporator, is evaporated and concentrated to one fourth of the volume of the stock solution, and then enters a crystallization tank for cooling and crystallization;
step 5) isolating crystals: centrifuging by using a centrifugal machine, increasing the rotating speed of the centrifugal machine from 450r/min to 950r/min at the speed of 10r/s, maintaining the rotating speed of 950r/min, centrifuging for about 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystals to obtain a powdery threonine product;
step 7) primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, centrifuging by a horizontal decanter centrifuge, and collecting crystals and secondary mother liquor;
step 8) chromatographic separation: performing chromatographic separation on the secondary mother liquor by using a sequential simulated moving bed, and removing impurities such as sugar, salt, pigment and the like to obtain a threonine-rich separation liquid;
step 9) crystallization and separation: crystallizing the separating liquid obtained in the step 8), centrifugally separating crystals, then combining the crystals with the crystals obtained in the step 7), and pumping the crystals into a crystallization tank in the step 4) for crystallization;
step 10) granulating: the powder threonine obtained in the step 6) is compressed into thin strips by a rolling granulator through adjusting the roller pressure and the roller spacing (the roller spacing is 2.2mm and the pressure is 200 kg), the pressure born by the material after extrusion forming is gradually reduced, and the material falls off downwards under the action of gravity and enters a crushing and granulating system; then the mixture enters a return auger according to the granularity requirement, passes through a sieve with 10 meshes and a sieve with 30 meshes in sequence, screens out coarse and fine particles, collects the particle products with the particle size of between 10 meshes and 30 meshes, and packages the products to obtain the finished product; the undersize powder is returned to granulation again.
Comparative example 1
A process for the production of granular threonine comprising the steps of:
step 1) fermentation: inoculating Escherichia coli engineering bacteria K12DeltadapA seed solution (inoculating Escherichia coli engineering bacteria into seed culture medium according to 10% inoculum size, culturing at 30deg.C and shaking table rotation speed of 180r/min until the concentration of seed solution is 1×10) 9 cfu/mL, the seed culture medium comprises the following components: 5g of yeast extract, 20g of glucose, 0.5g of ammonium sulfate, 0.1g of potassium dihydrogen sulfate, 0.01g of ferrous sulfate heptahydrate, 0.02g of magnesium sulfate heptahydrate, the balance of water and pH value of 6.5-7.0), inoculating the yeast extract into a fermentation tank containing a fermentation medium according to an inoculation amount of 6% to ferment, wherein the temperature is 30 ℃, the tank pressure is 0.04MPa, the ventilation amount is 0.6vvm, the rotating speed is 100rpm, the fermentation time is 72h, and the fermentation is stopped to collect fermentation liquor;
the components of the fermentation medium are as follows: glucose 50g/L, glycerin 20g/L, corn steep liquor 20g/L, ammonium sulfate 2g/L, monopotassium phosphate 0.2g/L, dipotassium phosphate 0.2g/L, magnesium sulfate heptahydrate 100mg/L, ferrous sulfate heptahydrate 10mg/L, manganese sulfate monohydrate 10mg/L, and pH value 6.5;
during the fermentation process:
1) Monitoring the concentration of glucose in the fermentation liquid, and controlling the sugar content to be 2% by feeding a first nutrient solution until the fermentation is finished, wherein the first nutrient solution comprises the following components: glucose 500g/L;
2) Monitoring the pH of the fermentation liquid, and controlling the pH to be 6.5 by adding 20% ammonia water until the fermentation is finished;
3) Adding purified water into the feed stream of the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquid after fermentation for about 24 hours;
step 2) centrifuging: centrifuging the fermentation liquor at 4500rpm for 5min by a disc centrifuge, and collecting supernatant liquid and precipitate;
step 3) membrane filtration: treating the upper liquid obtained in the step 2) by using a ceramic membrane, wherein the dialysate flow rate of a membrane assembly is 19m 3 And/h, the temperature of the membrane equipment is not higher than 80 ℃, the inlet pressure of the membrane is 0.3MPa, the outlet pressure of the membrane is 0.10MPa, the top pressure of the equipment is 0.15MPa, and the osmotic side pressure (high pressure side) is as follows: 0.15MPa, the permeate side pressure (low pressure side) is: 0.1MPa, the clear liquid flow (high pressure side) is: 15m 3 Flow rate of clear liquid (low pressure side): 10m 3 /h; the obtained ceramic membrane concentrated solution enters a horizontal decanter centrifuge for treatment, the rotating speed is 2000r/min, and the inlet flow is 16m 3 And (h) combining the supernatant obtained after centrifugation with a ceramic membrane permeate;
step 4) concentrating and crystallizing: then the mixture enters an evaporator, is evaporated and concentrated to one fourth of the volume of the stock solution, and then enters a crystallization tank for cooling and crystallization;
step 5) isolating crystals: centrifuging by using a centrifugal machine, increasing the rotation speed of the centrifugal machine from 400r/min to 900r/min at the speed of 10r/s, maintaining the rotation speed of 900r/min, centrifuging for about 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystals to obtain a powdery threonine product;
step 7) primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, centrifuging by a horizontal decanter centrifuge, and collecting crystals and secondary mother liquor;
step 8) chromatographic separation: performing chromatographic separation on the secondary mother liquor by using a sequential simulated moving bed, and removing impurities such as sugar, salt, pigment and the like to obtain a threonine-rich separation liquid;
step 9) crystallization and separation: crystallizing the separating liquid obtained in the step 8), centrifugally separating crystals, then combining the crystals with the crystals obtained in the step 7), and pumping the crystals into a crystallization tank in the step 4) for crystallization;
step 10) granulating: the powder threonine obtained in the step 6) is compressed into thin strips by a rolling granulator through adjusting the roller pressure and the roller spacing (the roller spacing is 2.2mm and the pressure is 175 kg), the pressure born by the material after extrusion forming is gradually reduced, and the material falls off downwards under the action of gravity and enters a crushing and granulating system; then the mixture enters a return auger according to the granularity requirement, passes through a sieve with 10 meshes and a sieve with 30 meshes in sequence, screens out coarse and fine particles, collects the particle products with the particle size of between 10 meshes and 30 meshes, and packages the products to obtain the finished product; the undersize powder is returned again to the granulation.
Example 3
Influence of different factors in the fermentation process on threonine content and sugar acid conversion rate in fermentation broth.
1. Optimizing on the basis of a conventional fermentation process comparative example 1, adding 4-hydroxy-3-methoxybenzoic acid with different concentrations into a fermentation medium, and setting concentration gradients as follows: 0,20,40,60,80,100,120,140 in mg/L, as shown in FIG. 1-2, the 4-hydroxy-3-methoxybenzoic acid is positively correlated with threonine content and sugar acid conversion rate, when the 4-hydroxy-3-methoxybenzoic acid concentration reaches 60mg/L, the peak value is close to that of the 4-hydroxy-3-methoxybenzoic acid, about 7 percent (threonine content) is increased compared with that of the non-added group, the 4-hydroxy-3-methoxybenzoic acid concentration (80 mg/L) is continuously increased, the threonine content and the sugar acid conversion rate are still slightly improved, and when the concentration reaches 100mg/L, both the threonine content and the sugar acid conversion rate synchronously fall back.
2. Based on the above work, the concentration of 4-hydroxy-3-methoxybenzoic acid in the fermentation medium was selected to be 80mg/L, and the effect of the first nutrient solution component (Table 1) on threonine content and sugar acid conversion rate was continuously verified, and the glucose concentration was 500g/L, with comparability.
TABLE 1
As shown in figure 3, methionine or isoleucine has a certain promotion effect on threonine content in fermentation broth, and when the methionine and the isoleucine are combined, the promotion effect is more obvious, methionine 2-3 g/L+isoleucine 2-3g/L has a more obvious promotion on threonine content, the addition amount is continuously increased, the influence on threonine content is small, and the sugar-acid conversion rate also shows a similar trend (not shown in the figure). In addition, the study also finds that the equal amount of methionine and isoleucine is fed in the early or middle fermentation stage, so that the influence on threonine yield is not great, and the possible reasons are that threonine synthesis is less in the early fermentation stage, the negative feedback mechanism of metabolic flow is weak, and a large amount of metabolic products are accumulated in the middle fermentation stage, so that certain negative feedback is caused.
3. Based on the above work, considering cost and other factors, the components of the first nutrient solution are selected as follows: glucose 500 g/L+methionine 2 g/L+isoleucine 2g/L, on the basis of which the optimization of the second nutrient solution component is continued, see in particular Table 2:
TABLE 2
Conclusion: metabolites such as acetic acid and the like can be produced in the middle and late growth period of the cells, so that not only is the carbon flux lost, but also the cells are damaged, and the cell activity is affected, the acetic acid synthesis pathway can be inhibited by feeding sodium acetate, the production of acetic acid is reduced, and the amount of converted ATP can be increased by providing a certain amount of adenosine, so that the strain aging too fast caused by insufficient intracellular ATP supply is avoided. According to the invention, the threonine yield is maximized by the compatibility mode of 20g/L sodium acetate and 5g/L adenosine through the compatibility proportion of sodium acetate and adenosine of different components as shown in figure 4.
In conclusion, the method adjusts a plurality of factors and metabolic points in the threonine synthesis pathway, optimizes the fermentation process to the greatest extent, and improves the fermentation efficiency and the sugar acid conversion rate.
The present invention is not limited to the above-mentioned embodiments, but is not limited to the above-mentioned embodiments, and any person skilled in the art will make some changes or modifications to the above-mentioned embodiments without departing from the scope of the present invention, but any simple modification, equivalent changes and modifications to the above-mentioned embodiments according to the technical matter of the present invention fall within the scope of the technical matter of the present invention.
Claims (8)
1. A process for the production of granular threonine, characterized in that it comprises the following steps:
step 1) fermentation: inoculating threonine producing escherichia coli engineering bacteria seed liquid into a fermentation tank containing a fermentation medium according to an inoculum size of 5-10% for fermentation, wherein the temperature is 30 ℃, the tank pressure is 0.04MPa, the ventilation amount is 0.6vvm, the rotating speed is 100rpm, the fermentation time is 72 hours, stopping fermentation, and collecting fermentation liquor;
the components of the fermentation medium are as follows: 50g/L of glucose, 20g/L of glycerol, 20g/L of corn steep liquor, 2g/L of ammonium sulfate, 0.2g/L of monopotassium phosphate, 0.2g/L of dipotassium phosphate, 100mg/L of magnesium sulfate heptahydrate, 60-80mg/L of 4-hydroxy-3-methoxybenzoic acid, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate and pH value of 6.5;
step 2) centrifuging: centrifuging the fermentation liquor at 4500-5000rpm for 4-5min by a disc centrifuge, and collecting supernatant liquid and precipitate;
step 3) membrane filtration: performing membrane filtration on the upper liquid obtained in the step 2) by utilizing a ceramic membrane, and collecting ceramic membrane concentrated solution and ceramic membrane permeate; the obtained ceramic membrane concentrated solution enters a horizontal decanter centrifuge for treatment, the rotating speed is 2000r/min, and the inlet flow is 16m 3 And (h) combining the supernatant obtained after centrifugation with a ceramic membrane permeate;
step 4) concentrating and crystallizing: entering an evaporator, evaporating and concentrating to one fourth of the volume of the stock solution, and then entering a crystallization tank for cooling and crystallization;
step 5) isolating crystals: centrifuging with a centrifuge, increasing the rotational speed of the centrifuge from 400-500r/min to 900-1000r/min at a speed of 10r/s, maintaining the rotational speed of 900-1000r/min, centrifuging for 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystals to obtain a powdery threonine product;
step 7) primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, centrifuging by a horizontal decanter centrifuge, and collecting crystals and secondary mother liquor;
step 8) chromatographic separation: performing chromatographic separation on the secondary mother liquor by using a sequential simulated moving bed to remove sugar, salt and pigment, thereby obtaining a threonine-rich separating liquid;
step 9) crystallization and separation: crystallizing the separating liquid obtained in the step 8), centrifugally separating crystals, then combining the crystals with the crystals obtained in the step 7), and pumping the crystals into a crystallization tank in the step 4) for crystallization;
step 10) granulating: compressing the powdery threonine obtained in the step 6) into a thin strip shape by using a granulator, and crushing and granulating; then sequentially passing through a 10-mesh sieve and a 30-mesh sieve, sieving to remove coarse and fine particles, collecting a particle product with the particle size of between 10 and 30 meshes, and packaging to obtain the product; the undersize powder is returned to the granulator again.
2. The process according to claim 1, wherein the fermentation of step 1) further comprises:
1) Monitoring the concentration of glucose in the fermentation liquid, and controlling the sugar content to be 2% by feeding the first nutrient solution until the fermentation is finished;
2) Monitoring the pH of the fermentation liquid, and controlling the pH to be 6.5 by adding 20% ammonia water until the fermentation is finished;
3) And (3) after fermentation for 24 hours, feeding a second nutrient solution into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquid until the fermentation is finished.
3. The production process according to claim 1, wherein the conditions of the membrane filtration are: membrane module dialysate flow 19m 3 And/h, the temperature of the membrane equipment is not higher than 80 ℃, the inlet pressure of the membrane is 0.3MPa, the outlet pressure of the membrane is 0.10MPa, the top pressure of the equipment is 0.15MPa, and the high pressure side of the osmotic side pressure is as follows: 0.15MPa, the low pressure side of the permeate side pressure is: the high pressure side of the clear liquid flow is 0.1 MPa: 15m 3 And/h, the low pressure side of the clear liquid flow is: 10m 3 /h。
4. The production process according to claim 1, wherein the roll pressure of the granulator is controlled to 175-200 kg and the roll spacing is controlled to 2.2-2.5mm.
5. The production process according to claim 2, wherein the first nutrient solution comprises the following components: glucose 500g/L, methionine 1-5g/L, isoleucine 1-5g/L.
6. The production process according to claim 2, wherein the second nutrient solution comprises the following components: 5-20g/L of sodium acetate and 1-5g/L of adenosine.
7. The production process according to claim 2, wherein the first nutrient solution comprises the following components: glucose 500g/L, methionine 2g/L, isoleucine 2g/L.
8. The production process according to claim 2, wherein the second nutrient solution comprises the following components: 20g/L of sodium acetate and 5g/L of adenosine.
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