CN112481321A - Production process of granular threonine - Google Patents
Production process of granular threonine Download PDFInfo
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- CN112481321A CN112481321A CN202010962335.7A CN202010962335A CN112481321A CN 112481321 A CN112481321 A CN 112481321A CN 202010962335 A CN202010962335 A CN 202010962335A CN 112481321 A CN112481321 A CN 112481321A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
- C07C227/40—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
- C07C227/40—Separation; Purification
- C07C227/42—Crystallisation
Abstract
The invention belongs to the technical field of amino acid production, and relates to a production process of granular threonine with insufficient happiness, which comprises the following steps: step 1) fermentation, step 2) centrifugation, step 3) membrane filtration, step 4) concentration and crystallization, step 5) crystal separation, 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 (Threonine, abbreviated as Thr) is known as 2-amino-3-hydroxybutyric acid, belongs to aliphatic amino acid, is slightly sweet, and has a structure similar to that of threose, and a molecule of the Threonine contains two asymmetric chiral carbon atoms, so that four stereoisomers exist. Among them, only L-threonine has biological activity. L-threonine is one of the eight essential amino acids with a relative molecular mass of 119.12, a melting point of 253 and 257 ℃. L-threonine is readily soluble in water and insoluble in chloroform, ethanol, diethyl ether, and the like.
L-threonine plays an important role in promoting the growth and development of the human body and maintaining normal physiological functions, and is therefore widely used in the fields of food and medicine. Nutritional supplements have an important role in the food industry because nutritional ingredients in food are easily destroyed and partially lost due to the current effects of large production volumes and wide distribution areas of food. Wherein, the threonine is used as an amino acid additive, can effectively enrich the nutrient contents and improve the oxidation resistance of the food. Meanwhile, the co-heating of threonine and glucose also plays a good role in increasing aroma. Secondly, threonine is also an essential amino acid for livestock and poultry. With methionine, lysine and tryptophan and is known as a four large amino acids feed additive. Threonine is added into the livestock and poultry feed, so that the amino acid types in the feed can be effectively enriched, the nitrogen utilization rate is improved, the protein synthesis is promoted, and the feed production cost is reduced. Researches show that the addition of threonine can effectively improve and maintain the feed intake and the immunocompetence of livestock and poultry and regulate the fat metabolism of animals. In addition, amino acids are in great demand in the medical field. Threonine is an important precursor for synthesizing various biological components in human body, and has the ability of improving immunity, so that threonine is widely used as one of components of amino acid infusion solution before and after operation and for adjuvant therapy of various diseases. In addition, iron threonine is also a widely used antianemia agent.
At present, three methods of threonine production are mainly fermentation, protein hydrolysis and chemical synthesis, and the microbial fermentation method has become the mainstream method of threonine production. The main production strains of L-threonine include Escherichia coli, Corynebacterium glutamicum, and Serratia marcescens. In addition, since Escherichia coli has advantages of easy cultivation, short fermentation time, clear genetic background, etc., Escherichia coli has been one of the most important strains for producing L-threonine in the fermentation industry so far.
The applicant has made a great deal of research on threonine fermentation. The literature is exemplified by the following:
"CN 110904167A, L-threonine fermentation process optimization method, 2020.3.24" discloses an L-threonine fermentation process optimization method, which comprises the following steps: inoculating the L-threonine production strain seed liquid into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 1-2% for fermentation, controlling the stirring speed at 300-37 ℃ and the dissolved oxygen at 500rpm, controlling the dissolved oxygen at 15-20% by aeration and stirring, defoaming by using a foam killer, and stopping fermentation for 36h, and collecting the fermentation liquid. The invention improves the yield of the L-threonine by optimizing the fermentation method. "CN 110894522A, a corn bran hydrolysate and its use in preparing threonine by fermentation, 2020.3.20" discloses a corn bran hydrolysate, which is prepared by the following process: step 1) corn bran pretreatment, step 2) ultrasonic-assisted hydrochloric acid hydrolysis, step 3) microbial hydrolysis, step 4) thallus hydrolysis, and step 5) concentration and steam treatment. The hydrolysate contains polypeptide, amino acid and reducing sugar, can be applied to threonine fermentation, reduces the cost and improves the fermentation efficiency.
"CN 110846348A, a method for preparing a threonine fermentation medium, 2020.02.28" discloses a method for preparing a threonine fermentation medium, which comprises the following steps: sequentially adding glucose, corn steep liquor, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, N-methyl aspartic acid, methionine, ferrous sulfate heptahydrate and manganese sulfate monohydrate into purified water, stirring for dissolving, and adjusting the pH value to 6.5-7.0. According to the invention, the fermentation medium is optimized, and 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.
"CN 110551773A, a method for using soybean meal enzymolysis liquid to replace yeast powder in threonine production, 2019.12.10", which comprises the following steps: 1) preparing bean pulp enzymolysis liquid, and 2) fermenting the bacterial strain. According to the invention, the soybean meal enzymolysis liquid is adopted to replace yeast extract in the culture medium, compared with the yeast extract, the yield and dry matter of threonine in the fermentation liquid are improved, and the cost is relatively low.
"CN 110396530A, a method for increasing threonine production and yield, 2019.11.1", discloses a method for increasing threonine production and yield, which comprises the following steps: step 1) preparing a fermentation medium, step 2) fermenting threonine, and step 3) separating, crystallizing and drying. The method of the invention reduces the cost and improves the yield and the yield.
"CN 110004192A, a method for preparing granule type threonine, 2019.7.12" discloses a method for preparing granule type threonine, which comprises the following steps: step 1) fermenting, step 2) centrifuging, and step 3) preparing granular threonine. The method can improve the content of the granule threonine, and has simple and feasible process and wide application prospect.
"CN 109609566A, a method for increasing threonine production, 2019.04.12" discloses a method for increasing threonine production, which comprises the following steps: inoculating the threonine-producing engineering bacteria seed liquid of escherichia coli into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 10-12% for fermentation, wherein the temperature is 30-34 ℃, the tank pressure is 0.03-0.04MPa, the ventilation volume is 0.4-0.6vvm, the rotating speed is 100rpm, the fermentation time is 30-36h, then adding 200mg/L of inositol, continuing the fermentation culture for 24-30h, stopping the fermentation, and collecting the fermentation liquid. The invention improves the yield of threonine by optimizing the fermentation process.
Other relevant institutions have also studied, for example:
"Jilin university CN107760734A, a method for improving threonine production and application thereof, 2018.03.06" discloses a method for improving threonine production, wherein a mixed fermentation accelerator consisting of betaine hydrochloride, VB4 and VB3 is added in an L-threonine fermentation process, so that the L-threonine production can be improved by 10-12%.
"Tianjin science and technology university CN107012181A, a threonine fermentation medium and threonine clean production process, 2017.08.04" discloses a threonine fermentation medium and threonine clean production process, wherein the fermentation medium does not contain corn steep liquor and yeast powder, and achieves substitution effect by adding various amino acids, nucleosides and microelements, and the medium is used for threonine clear liquid fermentation to improve saccharic acid conversion rate and extraction yield.
By analysis, the traditional threonine production process mainly has the following defects: the fermentation yield is low, and the fermentation process is not comprehensively and systematically optimized; traditional powdered threonine is unfavorable for transportation and storage because the granularity is less, absorbs moisture easily and agglomerates, and in the feed addition use, the mobility is poor, and the dust is big, is unfavorable for adding, has brought certain restriction for the development of threonine trade. It is necessary to develop a novel process for producing threonine in the form of granules to compensate for the above-mentioned disadvantages.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a production process of granular threonine.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a process for the production of particulate threonine comprising the steps of: step 1) fermentation, step 2) centrifugation, step 3) membrane filtration, step 4) concentration and crystallization, step 5) crystal separation, 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 amount of the solvent to be used,
the production process comprises the following steps:
step 1) fermentation: inoculating the threonine-producing engineering bacteria seed liquid of escherichia coli into a fermentation tank containing a fermentation medium according to the inoculation amount of 6% for fermentation, stopping fermentation and collecting fermentation liquid, wherein the temperature is 30 ℃, the tank pressure is 0.04MPa, the ventilation volume is 0.6vvm, the rotating speed is 100rpm, and the fermentation time is 72 hours;
step 2) centrifugation: the fermentation liquid is firstly centrifuged for 4-5min at 4500-;
step 3) membrane filtration: membrane filtering the upper layer liquid obtained in the step 2) by using a ceramic membrane, and collecting a ceramic membrane concentrated solution and a ceramic membrane permeate; the obtained ceramic membrane concentrated solution enters a horizontal screw centrifuge for processing, the rotating speed is 2000r/min, and the machine inlet flow is 16m3H, mixing the supernatant obtained after centrifugation with the ceramic membrane permeate to obtain a combined permeate;
step 4), concentration and crystallization: feeding the combined permeate obtained in the step 3) into an evaporator, evaporating and concentrating to one fourth of the volume of the stock solution, and then feeding into a crystallization tank for cooling and crystallization;
step 5) crystal separation: centrifuging by a centrifuge, wherein the rotating speed of the centrifuge is increased from 400-500r/min to 900-1000r/min at the speed of 10r/s, then maintaining the rotating speed of 900-1000r/min for centrifuging for 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystal to obtain a powdery threonine product;
step 7), primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, then centrifuging by a horizontal screw 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 to obtain a threonine-rich separation solution;
step 9) crystallization and separation: crystallizing the separation liquid obtained in the step 8), centrifugally separating crystals, combining the crystals with the crystals obtained in the step 7), and pumping the combined crystals into a crystallizing tank in the step 4) for crystallization;
step 10) granulation: compressing the powdery threonine obtained in the step 6) into a fine strip shape by using a rolling granulator, gradually reducing the pressure borne by the material after extrusion forming, falling off downwards under the action of gravity, and entering a crushing and granulating system; then feeding the mixture into a feed back auger according to the granularity requirement, sequentially sieving the mixture through a 10-mesh sieve and a 30-mesh sieve to remove coarse and fine particles, collecting particle products with the particle size of between 10 meshes and 30 meshes, and packaging to obtain the feed back auger; the undersize powder is returned to the granulation.
Further, 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 6.5 of pH value.
Further, the air conditioner is provided with a fan,
the step 1) of fermentation further comprises the following steps:
1) monitoring the glucose concentration in the fermentation liquor, and controlling the sugar content to be 2% by feeding the first nutrient solution until the fermentation is finished;
2) monitoring the pH value of the fermentation liquor, and controlling the pH value to be 6.5 by adding 20% ammonia water in a flowing manner until the fermentation is finished;
3) and feeding the second nutrient solution into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquor after the fermentation is carried out for about 24h until the fermentation is finished.
Further, the air conditioner is provided with a fan,
the conditions of the membrane filtration are as follows: flow rate of membrane module dialysate 19m3The temperature of the membrane equipment is not higher than 80 ℃, the pressure at the inlet of the membrane is 0.3MPa, the pressure at the outlet of the membrane is 0.10MPa, the pressure at the top of the equipment is 0.15MPa, and the pressure at the permeation side (high pressure side) is as follows: 0.15MPa, permeate side pressure (low pressure side): 0.1MPa, clear solution flow (high pressure side): 15m3H, clear liquid flow (low pressure side): 10m3/h。
Further, the roller pressure of the rolling granulator is controlled at 175-200 kg, and the roller spacing is controlled at 2.2-2.5 mm.
Further, the air conditioner is provided with a fan,
the first nutrient solution comprises the following components: 500g/L glucose, 1-5g/L methionine and 1-5g/L isoleucine.
Further, the air conditioner is provided with a fan,
the second nutrient solution comprises the following components: 5-20g/L of sodium acetate and 1-5g/L of adenosine.
Further, in the present invention,
the first nutrient solution comprises the following components: glucose 500g/L, methionine 2g/L, isoleucine 2 g/L.
Further, in the present invention,
the second nutrient solution comprises the following components: sodium acetate 20g/L + adenosine 5 g/L.
The starting point and the beneficial effects of the research of the invention mainly comprise but are not limited to the following aspects:
the pentose phosphate pathway provides glyceraldehyde triphosphate to enter a threonine synthesis pathway, and also can provide a large amount of NADPH for threonine synthesis, so that the synthesis of threonine is promoted; according to the invention, 4-hydroxy-3-methoxybenzoic acid is added into the fermentation medium, so that various key enzymes in the pentose phosphate pathway, including glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, can be activated, the pentose phosphate pathway is enhanced, and the sugar acid conversion rate and the fermentation efficiency of threonine are further improved.
The methionine and lysine synthesis pathways can generate a shunt for threonine synthesis, and as the yield of threonine is increased, the downstream product isoleucine begins to accumulate, so that the carbon flux is lost, and the increase of the threonine metabolic flux can be promoted by reducing the metabolic flux entering the methionine and lysine pathways; the K12dapA escherichia coli belongs to a lysine-producing defective strain, and the lysine pathway is not required to be regulated and controlled; when glucose is consumed to be below 2%, the strain proliferation is slowed down, metabolites are synthesized in a large amount, glucose, methionine and isoleucine are supplemented, glucose is supplemented with sugar for metabolic use of the strain, methionine and isoleucine can generate a certain feedback inhibition effect on methionine and isoleucine, and the normal metabolism and proliferation of the strain are not influenced.
Metabolites such as acetic acid and the like can be generated in the middle and later stages of cell growth, so that carbon flow loss is caused, the cells are damaged, the cell activity is influenced, the acetic acid synthesis path can be inhibited by adding sodium acetate in a flowing manner, the generation of acetic acid is reduced, the amount of converted ATP can be increased by providing a certain amount of adenosine, and the condition that the strain is aged too fast due to insufficient supply of intracellular ATP is avoided.
The invention aims at adjusting a plurality of factors and metabolic points in the threonine synthesis pathway, and aims at optimizing the fermentation process to the maximum extent and improving the fermentation efficiency and the sugar-acid conversion rate.
In the invention, powdery threonine is prepared into granular threonine, and a disc centrifuge is adopted for centrifugal treatment, so that the pressure of membrane separation is reduced; the invention prepares the threonine into the granular threonine, can improve the fluidity, is convenient for storage and transportation, controls the solubility, improves the quality and the added value of the threonine and increases the economic value.
Drawings
FIG. 1: influence of the concentration of 4-hydroxy-3-methoxybenzoic acid on the threonine content of the fermentation broth;
FIG. 2: influence of 4-hydroxy-3-methoxybenzoic acid concentration on the conversion rate of sugar and acid;
FIG. 3: the effect of the first nutrient solution component on the threonine content of the fermentation broth;
FIG. 4: the effect of the second nutrient solution component on the threonine content of the fermentation broth.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
A process for the production of particulate threonine comprising the steps of:
step 1) fermentation: inoculating Escherichia coli engineering bacteria K12 delta dapA seed solution (preparation method comprises inoculating 10% of Escherichia coli engineering bacteria into seed culture medium, culturing at 30 deg.C and shaking table rotation speed of 180r/min until the concentration of seed solution is1×109cfu/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 and the balance of water, wherein the pH value is 6.5-7.0) is inoculated into a fermentation tank containing a fermentation medium according to the inoculation amount of 6% for fermentation, 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, the fermentation is stopped, and the fermentation broth is collected;
the fermentation medium comprises the following components: 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, 80mg/L of 4-hydroxy-3-methoxybenzoic acid, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate and the pH value of 6.5; in the fermentation process:
1) monitoring the glucose concentration in the fermentation liquor, 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: 500g/L glucose, 2g/L methionine and 2g/L isoleucine;
2) monitoring the pH value of the fermentation liquor, and controlling the pH value to be 6.5 by adding 20% ammonia water in a flowing manner until the fermentation is finished;
3) feeding a second nutrient solution into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquor after fermentation is carried out for about 24h 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) centrifugation: centrifuging the fermentation liquor by a disc centrifuge at 4500rpm for 5min, and collecting upper layer liquid and precipitate;
step 3) membrane filtration: treating the upper layer liquid obtained in the step 2) by using a ceramic membrane, wherein the flow of the membrane module dialysate is 19m3The temperature of the membrane equipment is not higher than 80 ℃, the pressure at the inlet of the membrane is 0.3MPa, the pressure at the outlet of the membrane is 0.10MPa, the pressure at the top of the equipment is 0.15MPa, and the pressure at the permeation side (high pressure side) is as follows: 0.15MPa, permeate side pressure (low pressure side): 0.1MPa, clear solution flow (high pressure side): 15m3H, clear liquid flow (low pressure side): 10m3H; the obtained ceramic membrane concentrated solution enters a horizontal screw centrifuge for processing, the rotating speed is 2000r/min, and the machine inlet flow is 16m3H, mixing the supernatant obtained after centrifugation with the ceramic membrane permeateAnd;
step 4), concentration and crystallization: the combined solution obtained in the step 3) enters an evaporator, is evaporated and concentrated to one fourth of the volume of the original solution, and then enters a crystallizing tank for cooling and crystallizing;
step 5) crystal separation: centrifuging by a centrifuge, increasing the rotating speed of the centrifuge from 400r/min to 900r/min at the speed of 10r/s, then maintaining the rotating speed of 900r/min for centrifuging for about 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystal to obtain a powdery threonine product;
step 7), primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, then centrifuging by a horizontal screw 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 impurities such as sugar, salt, pigment and the like to obtain a separation liquid rich in threonine;
step 9) crystallization and separation: crystallizing the separation liquid obtained in the step 8), centrifugally separating crystals, combining the crystals with the crystals obtained in the step 7), and pumping the combined crystals into a crystallizing tank in the step 4) for crystallization;
step 10) granulation: compressing the powdery threonine obtained in the step 6) into a fine strip shape by using a rolling granulator and adjusting the roller pressure and the roller spacing (the roller spacing is 2.2mm, and the pressure is 175 kg), wherein the pressure borne by the material after extrusion forming is gradually reduced, the material falls downwards under the action of gravity, and the material enters a crushing and granulating system; then feeding the mixture into a feed back auger according to the granularity requirement, sequentially sieving the mixture through a 10-mesh sieve and a 30-mesh sieve to remove coarse and fine particles, collecting particle products with the particle size of between 10 meshes and 30 meshes, and packaging to obtain the feed back auger; the undersize powder is returned to the granulation.
Example 2
A process for the production of particulate threonine comprising the steps of:
step 1) fermentation: inoculating Escherichia coli engineering bacteria K12 delta dapA seed solution (inoculating Escherichia coli engineering bacteria into seed culture medium according to 10% inoculum size, culturing at 30 deg.C and shaking table rotation speed of 180r/minUntil the concentration of the seed liquid is 1 x 109cfu/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 and the balance of water, wherein the pH value is 6.5-7.0) is inoculated into a fermentation tank containing a fermentation medium according to the inoculation amount of 6% for fermentation, 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, the fermentation is stopped, and the fermentation broth is collected;
the fermentation medium comprises the following components: 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, 60mg/L of 4-hydroxy-3-methoxybenzoic acid, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate and the pH value of 6.5; in the fermentation process:
1) monitoring the glucose concentration in the fermentation liquor, 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: 500g/L glucose, 3g/L methionine and 3g/L isoleucine;
2) monitoring the pH value of the fermentation liquor, and controlling the pH value to be 6.5 by adding 20% ammonia water in a flowing manner until the fermentation is finished;
3) feeding a second nutrient solution into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquor after fermentation is carried out for about 24h until the fermentation is finished, wherein the second nutrient solution comprises the following components: sodium acetate 15g/L + adenosine 4 g/L; step 2) centrifugation: centrifuging the fermentation liquor by a disc centrifuge at 4500rpm for 5min, and collecting upper layer liquid and precipitate;
step 3) membrane filtration: treating the upper layer liquid obtained in the step 2) by using a ceramic membrane, wherein the flow of the membrane module dialysate is 19m3The temperature of the membrane equipment is not higher than 80 ℃, the pressure at the inlet of the membrane is 0.3MPa, the pressure at the outlet of the membrane is 0.10MPa, the pressure at the top of the equipment is 0.15MPa, and the pressure at the permeation side (high pressure side) is as follows: 0.15MPa, permeate side pressure (low pressure side): 0.1MPa, clear solution flow (high pressure side): 15m3H, clear liquid flow (low pressure side): 10m3H; the obtained ceramic membrane concentrated solution enters a horizontal screw centrifuge for processing, the rotating speed is 2000r/min, and the machine inlet flow is 16m3H, centrifuging the resulting supernatant andcombining the ceramic membrane permeate;
step 4), concentration and crystallization: then 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 crystallizing;
step 5) crystal separation: centrifuging by a centrifuge, increasing the rotating speed of the centrifuge from 450r/min to 950r/min at the speed of 10r/s, then maintaining the rotating speed of 950r/min for centrifuging for about 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystal to obtain a powdery threonine product;
step 7), primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, then centrifuging by a horizontal screw 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 impurities such as sugar, salt, pigment and the like to obtain a separation liquid rich in threonine;
step 9) crystallization and separation: crystallizing the separation liquid obtained in the step 8), centrifugally separating crystals, combining the crystals with the crystals obtained in the step 7), and pumping the combined crystals into a crystallizing tank in the step 4) for crystallization;
step 10) granulation: compressing the powdery threonine obtained in the step 6) into a fine strip shape by using a rolling granulator and adjusting the roller pressure and the roller spacing (the roller spacing is 2.2mm, and the pressure is 200 kg), wherein the pressure borne by the material after extrusion forming is gradually reduced, the material falls downwards under the action of gravity, and the material enters a crushing and granulating system; then feeding the mixture into a feed back auger according to the granularity requirement, sequentially sieving the mixture through a 10-mesh sieve and a 30-mesh sieve to remove coarse and fine particles, collecting particle products with the particle size of between 10 meshes and 30 meshes, and packaging to obtain the feed back auger; the sieved powder is returned to granulation again.
Comparative example 1
A process for the production of particulate threonine comprising the steps of:
step 1) fermentation: inoculating Escherichia coli engineering bacteria K12 delta dapA seed solution (inoculating Escherichia coli engineering bacteria into seed culture medium according to 10% inoculum size, culturing at 30 deg.C and shaking table rotation speed of 180r/min,culturing until the concentration of seed liquid is 1 × 109cfu/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 and the balance of water, wherein the pH value is 6.5-7.0) is inoculated into a fermentation tank containing a fermentation medium according to the inoculation amount of 6% for fermentation, 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, the fermentation is stopped, and the fermentation broth is collected;
the fermentation medium comprises the following components: 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, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate and 6.5 of pH value;
in the fermentation process:
1) monitoring the glucose concentration in the fermentation liquor, 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: 500g/L of glucose;
2) monitoring the pH value of the fermentation liquor, and controlling the pH value to be 6.5 by adding 20% ammonia water in a flowing manner until the fermentation is finished;
3) after fermenting for about 24 hours, adding purified water into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquor;
step 2) centrifugation: centrifuging the fermentation liquor by a disc centrifuge at 4500rpm for 5min, and collecting upper layer liquid and precipitate;
step 3) membrane filtration: treating the upper layer liquid obtained in the step 2) by using a ceramic membrane, wherein the flow of the membrane module dialysate is 19m3The temperature of the membrane equipment is not higher than 80 ℃, the pressure at the inlet of the membrane is 0.3MPa, the pressure at the outlet of the membrane is 0.10MPa, the pressure at the top of the equipment is 0.15MPa, and the pressure at the permeation side (high pressure side) is as follows: 0.15MPa, permeate side pressure (low pressure side): 0.1MPa, clear solution flow (high pressure side): 15m3H, clear liquid flow (low pressure side): 10m3H; the obtained ceramic membrane concentrated solution enters a horizontal screw centrifuge for processing, the rotating speed is 2000r/min, and the machine inlet flow is 16m3H, mixing the supernatant obtained after centrifugation with the ceramic membrane permeate;
step 4), concentration and crystallization: then 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 crystallizing;
step 5) crystal separation: centrifuging by a centrifuge, increasing the rotating speed of the centrifuge from 400r/min to 900r/min at the speed of 10r/s, then maintaining the rotating speed of 900r/min for centrifuging for about 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystal to obtain a powdery threonine product;
step 7), primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, then centrifuging by a horizontal screw 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 impurities such as sugar, salt, pigment and the like to obtain a separation liquid rich in threonine;
step 9) crystallization and separation: crystallizing the separation liquid obtained in the step 8), centrifugally separating crystals, combining the crystals with the crystals obtained in the step 7), and pumping the combined crystals into a crystallizing tank in the step 4) for crystallization;
step 10) granulation: compressing the powdery threonine obtained in the step 6) into a fine strip shape by using a rolling granulator and adjusting the roller pressure and the roller spacing (the roller spacing is 2.2mm, and the pressure is 175 kg), wherein the pressure borne by the material after extrusion forming is gradually reduced, the material falls downwards under the action of gravity, and the material enters a crushing and granulating system; then feeding the mixture into a feed back auger according to the granularity requirement, sequentially sieving the mixture through a 10-mesh sieve and a 30-mesh sieve to remove coarse and fine particles, collecting particle products with the particle size of between 10 meshes and 30 meshes, and packaging to obtain the feed back auger; the undersize powder is returned to the granulation.
Example 3
Influence of different factors in the fermentation process on the threonine content and the sugar acid conversion rate in the fermentation liquid.
1. Optimizing on the basis of the comparative example 1 of the conventional fermentation process, adding 4-hydroxy-3-methoxybenzoic acid with different concentrations into a fermentation culture medium, and setting concentration gradients as follows: 0,20,40,60,80,100,120 and 140, wherein the unit is mg/L, as shown in figure 1-2, 4-hydroxy-3-methoxybenzoic acid is positively correlated with threonine content and sugar acid conversion rate, when the concentration of 4-hydroxy-3-methoxybenzoic acid reaches 60mg/L, the peak value is approached, the concentration is increased by about 7 percent (threonine content) compared with the non-added group, the concentration of 4-hydroxy-3-methoxybenzoic acid (80mg/L) is continuously increased, small promotion still exists, and when the concentration reaches 100mg/L, the threonine content and the sugar acid conversion rate are both synchronously fallen back.
2. On the basis of the work, the concentration of the 4-hydroxy-3-methoxybenzoic acid in the selected fermentation medium is 80mg/L, the influence of the first nutrient solution component (shown in table 1) on the threonine content and the saccharic acid conversion rate is continuously verified, and the glucose concentration is 500g/L, so that the comparative performance is achieved.
TABLE 1
As shown in figure 3, methionine or isoleucine has a certain promotion effect on threonine content in the fermentation broth, and the promotion effect is more obvious when the methionine and the isoleucine are used in combination, the threonine content is obviously promoted when the methionine is 2-3g/L and the isoleucine is 2-3g/L, the addition amount is increased continuously, the influence on the threonine content is not large, and the sugar-acid conversion rate shows a similar trend (not shown in figure). In addition, the research also finds that the influence on the yield of threonine is not great when equal amounts of methionine and isoleucine are fed in the early fermentation stage or the middle fermentation stage, probably because threonine synthesis is less in the early fermentation stage, the negative feedback mechanism of metabolic flux is weak, and a large amount of metabolites are accumulated in the middle fermentation stage, so that certain negative feedback is caused.
3. On the basis of the work, considering factors such as cost and the like, the components of the first nutrient solution are selected as follows: glucose 500g/L + methionine 2g/L + isoleucine 2g/L, on this basis, the second nutrient solution components were continuously optimized, as shown in Table 2:
TABLE 2
And (4) conclusion: metabolites such as acetic acid and the like can be generated in the middle and later stages of cell growth, so that carbon flow loss is caused, the cells are damaged, the cell activity is influenced, the acetic acid synthesis path can be inhibited by adding sodium acetate in a flowing manner, the generation of acetic acid is reduced, the amount of converted ATP can be increased by providing a certain amount of adenosine, and the condition that the strain is aged too fast due to insufficient supply of intracellular ATP is avoided. According to the invention, the yield of threonine is increased to the maximum extent by the compatibility of sodium acetate and adenosine with different components, as shown in figure 4, and the compatibility of 20g/L sodium acetate and 5g/L adenosine.
In conclusion, the invention adjusts a plurality of factors and metabolic points in the threonine synthesis pathway, optimizes the fermentation process to the maximum extent, and improves the fermentation efficiency and the sugar-acid conversion rate.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. 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 and crystallization, step 5) crystal separation, step 6) drying, step 7) primary mother liquor crystallization and separation, step 8) chromatographic separation, step 9) crystallization and separation, and step 10) granulation.
2. The production process according to claim 1, characterized in that it comprises the following steps:
step 1) fermentation: inoculating the threonine-producing engineering bacteria seed liquid of escherichia coli into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 5-10% for fermentation, wherein the temperature is 30 ℃, the tank pressure is 0.04MPa, the ventilation volume is 0.6vvm, the rotating speed is 100rpm, the fermentation time is 72h, stopping fermentation, and collecting fermentation liquor;
step 2) centrifugation: the fermentation liquid is firstly centrifuged for 4-5min at 4500-;
step 3) membrane filtration: membrane filtering the upper layer liquid obtained in the step 2) by using a ceramic membrane, and collecting a ceramic membrane concentrated solution and a ceramic membrane permeate; the obtained ceramic membrane concentrated solution enters a horizontal screw centrifuge for processing, the rotating speed is 2000r/min, and the machine inlet flow is 16m3H, mixing the supernatant obtained after centrifugation with the ceramic membrane permeate;
step 4), concentration and crystallization: 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 crystallizing;
step 5) crystal separation: centrifuging by a centrifuge, wherein the rotating speed of the centrifuge is increased from 400-500r/min to 900-1000r/min at the speed of 10r/s, then maintaining the rotating speed of 900-1000r/min for centrifuging for 150s, and collecting crystals and primary mother liquor;
step 6) drying: drying the separated crystal to obtain a powdery threonine product;
step 7), primary mother liquor crystallization and separation: concentrating and crystallizing the primary mother liquor, then centrifuging by a horizontal screw 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 to obtain a threonine-rich separation solution;
step 9) crystallization and separation: crystallizing the separation liquid obtained in the step 8), centrifugally separating crystals, combining the crystals with the crystals obtained in the step 7), and pumping the combined crystals into a crystallizing tank in the step 4) for crystallization;
step 10) granulation: compressing the powdery threonine obtained in the step 6) into a fine strip shape by using a granulator, and crushing and granulating the fine strip shape; then sequentially passing through a 10-mesh sieve and a 30-mesh sieve, removing coarse and fine particles, collecting particle products with the particle size of 10-30 meshes, and packaging to obtain the product; the sieved powder is returned to the granulator again.
3. The production process according to claim 2, wherein the fermentation medium has the composition: 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 6.5 of pH value.
4. The process of claim 2, wherein step 1) fermentation further comprises:
1) monitoring the glucose concentration in the fermentation liquor, and controlling the sugar content to be 2% by feeding the first nutrient solution until the fermentation is finished;
2) monitoring the pH value of the fermentation liquor, and controlling the pH value to be 6.5 by adding 20% ammonia water in a flowing manner until the fermentation is finished;
3) and after fermentation is carried out for about 24 hours, feeding the second nutrient solution into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquor until the fermentation is finished.
5. The production process according to claim 2, wherein the conditions of the membrane filtration are: flow rate of membrane module dialysate 19m3The temperature of the membrane equipment is not higher than 80 ℃, the pressure at the inlet of the membrane is 0.3MPa, the pressure at the outlet of the membrane is 0.10MPa, the pressure at the top of the equipment is 0.15MPa, and the pressure at the high pressure side of the permeation side is as follows: 0.15MPa, the low pressure side of the permeate side pressure is: 0.1MPa, the high pressure side of the clear liquid flow is: 15m3The low pressure side of the clear liquid flow is: 10m3/h。
6. The production process according to claim 2, wherein the pressure of the rollers of the granulator is controlled to be 175-200 kg, and the distance between the rollers is controlled to be 2.2-2.5 mm.
7. The production process according to claim 4, wherein the first nutrient solution comprises the following components: 500g/L glucose, 1-5g/L methionine and 1-5g/L isoleucine.
8. The process according to claim 4, wherein the second nutrient solution comprises the following components: 5-20g/L of sodium acetate and 1-5g/L of adenosine.
9. The production process according to claim 7, wherein the first nutrient solution comprises the following components: glucose 500g/L, methionine 2g/L, isoleucine 2 g/L.
10. The process according to claim 8, wherein the second nutrient solution comprises the following components: sodium acetate 20g/L + adenosine 5 g/L.
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