CN110607331A - Process for preparing and extracting L-leucine - Google Patents
Process for preparing and extracting L-leucine Download PDFInfo
- Publication number
- CN110607331A CN110607331A CN201911003541.9A CN201911003541A CN110607331A CN 110607331 A CN110607331 A CN 110607331A CN 201911003541 A CN201911003541 A CN 201911003541A CN 110607331 A CN110607331 A CN 110607331A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- concentration
- membrane
- decolorization
- leucine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the field of L-leucine production processes, and discloses a process for preparing and extracting L-leucine, which comprises the following steps: step 1) fermentation, step 2) centrifugation, step 3) ceramic membrane filtration, step 4) activated carbon decolorization, step 5) decolorization membrane decolorization, step 6) sequential simulated moving bed chromatography extraction, and step 7) concentration, centrifugation and drying. The process has the advantages of high fermentation efficiency, energy conservation, emission reduction, and high product yield and purity.
Description
Technical Field
The invention belongs to the field of L-leucine production processes, and particularly provides a process for preparing and extracting L-leucine.
Background
L-leucine is also called leucine, its chemical name is alpha-aminoisocaproic acid, and its molecular formula is C6H13O2N, relative molecular weight 131.18, is a nonpolar amino acid, slightly bitter in taste, soluble in water, has an isoelectric point of 5.98, and has wide application in many industries such as the manufacture of medicines, foods, flavoring agents, animal feeds, cosmetics, and the like. The research of China on L-leucine starts relatively late, the L-leucine production level of partial enterprises by fermentation methods is relatively low, the research on downstream pretreatment and extraction processes is relatively less, the industrial production is difficult to realize, and the problems of high acid and alkali consumption, serious pollution, low product yield and purity and the like exist.
At present, the production method of L-leucine is mainly a fermentation method, and the L-leucine fermentation liquor produced by the fermentation method can be prepared into a finished product through the steps of extraction, evaporation concentration, centrifugal drying and the like. The prior patent technology of the applicant, namely 'a fermentation process for producing L-leucine', improves the yield of leucine by feeding a compound amino acid solution and a nutrient solution in the middle and later stages of fermentation, but the feeding solution has higher cost and is not suitable for large-scale production. The prior patent technology of the applicant 'a method for improving the fermentation yield of L-leucine', membrane coupling intermittent dialysis fermentation is adopted, and byproducts are effectively reducedIs/are as followsThe concentration improves the sugar acid conversion rate, and the L-leucine yield and the sugar acid conversion rate in the fermentation liquid can be promoted by supplementing the nutrient solution containing the glycerol and the betaine in the middle fermentation period; however, this method has a drawback of complicated steps. The technical problem to be solved is to provide a leucine fermentation process which is low in cost, easy to operate and capable of being developed sustainably.
The existing extraction method of L-leucine generally adopts a sedimentation method, an ion exchange method, a full-membrane method and the like, is limited in separation, has large wastewater production amount, causes large pressure for environmental protection, is easy to realize automation of chromatographic separation, does not need regeneration, has low energy consumption, high separation efficiency and strong adaptability, ensures that the purity of leucine in a collected liquid is up to more than 95 percent after the chromatographic separation of a simulated moving bed, has less sewage discharge than 1/3 of the traditional process, and is easy to treat.
Disclosure of Invention
In order to solve the common problem of industrial preparation and extraction of L-leucine, the invention provides a process for preparing and extracting L-leucine, which has the advantages of high fermentation efficiency, energy conservation, emission reduction, high product yield and high purity.
The invention is realized by the following technical scheme.
A process for preparing and extracting L-leucine, comprising the steps of:
step 1) fermentation, step 2) centrifugation, step 3) ceramic membrane filtration, step 4) activated carbon decolorization, step 5) decolorization membrane decolorization, step 6) sequential simulated moving bed chromatography extraction, and step 7) concentration, centrifugation and drying.
Further, the process comprises the following steps:
step 1) fermentation: inoculating the L-leucine-producing Brevibacterium flavum seed solution into a fermentation tank filled with a fermentation tank culture medium according to the inoculation amount of 8-10%, and stirring at the rotation speed of 300-; controlling the dissolved oxygen to be 20-30% by stirring and ventilating; controlling the pH value to be 7.0 by adding ammonia water in a flowing manner; the culture temperature is 33 ℃; defoaming with foam killer; when the fermentation time is 20 hours, feeding nutrient solution into the fermentation tank, maintaining the sugar concentration in the fermentation tank to be 1g/L, and stopping feeding 6 hours before the fermentation is finished; the fermentation period is 44 h;
step 2) centrifugation: taking the fermentation liquid, centrifuging for 3-5min at 4000-;
step 3) ceramic membrane filtration: performing primary filtration treatment on the fermentation liquor in the filter liquor obtained in the step 2) in a ceramic membrane of a multistage membrane coupling system, and collecting the filtered clear liquor;
step 4), activated carbon decolorization: performing active carbon decolorization treatment on the filtered clear liquid by using a plate-type closed filter, wherein the decolorization time is 60-65 min;
step 5) decoloring by a decoloring membrane: performing secondary decolorization treatment on the decolorized clear liquid in a decolorizing membrane of a multistage membrane coupling system to obtain decolorized clear liquid;
step 6) sequential simulated moving bed chromatographic extraction: evaporating and concentrating the decolorized clear liquid, and performing three-stage extraction treatment in a sequential simulated moving bed chromatography of a multistage membrane coupling system to obtain an extracted clear liquid;
step 7) concentrating, centrifuging and drying: and (4) carrying out four-effect concentration, centrifugation, fluidized bed drying and screen packaging on the extracted clear liquid to obtain the high-purity L-leucine.
Further, the air conditioner is provided with a fan,
when the fermentation is carried out for 30 hours, adding a sodium acetate aqueous solution into the fermentation tank, and controlling the concentration of sodium acetate in the fermentation liquor to be 0.1-1g/L until the fermentation is finished, wherein the concentration of the sodium acetate aqueous solution is 50-100 g/L;
when the fermentation time is 30 hours, adding a malonic acid aqueous solution into the fermentation tank, and controlling the concentration of malonic acid in the fermentation liquid to be 0.1-0.5 ml/L until the fermentation is finished; the concentration of the malonic acid aqueous solution is 10-20%;
when the fermentation time is up to 40h, adding chitosan into the fermentation tank, and controlling the concentration of the chitosan in the fermentation liquor to be 20-80 mg/L.
Preferably, the components of the fermenter medium: glucose 50g/L, (NH)4)2SO410g/L of corn steep liquor, 10g/L of KH2PO4 4g/L，MgSO4·7H2O1.5 g/L, citric acid 0.5g/L, MnSO4·H2O 10mg/L，FeSO4·7H2O 0.1g/L，VB15mg/L, biotin 50. mu.g/L.
Preferably, the components of the nutrient solution are: 100g/L of glucose, 5g/L of glycerol and 1g/L of betaine.
Preferably, the concentration of sodium acetate in the fermentation liquor is controlled to be 0.4-0.5 g/L; controlling the concentration of malonic acid in the fermentation liquor to be 0.2-0.3 ml/L; the concentration of chitosan in the fermentation liquor is controlled to be 40-50 mg/L.
Preferably, the ceramic membrane filtration parameters are: the pressure difference is 0.06-0.07MPa, the temperature is 50 ℃, and the pH value is 6.0-6.5.
Preferably, the decolorization parameters of the decolorizing membrane are as follows: the membrane inlet pressure is 0.9MPa, the membrane outlet pressure is 0.8MPa, and the flow rate of the feed liquid is 120 mL/min.
Preferably, the parameters of the sequential simulated moving bed chromatographic extraction are as follows: pH5.0, flow rate of column feeding 0.7BV/h, and elution speed 0.7 BV/h.
Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:
in the fermentation process, acetic acid is one of main byproducts in the fermentation process of leucine; the invention discovers that in the fermentation process, the sodium acetate is added, so that the feedback inhibition can be carried out on the acetic acid synthetic pathway, the acetic acid synthetic pathway is blocked, the generation amount of Val is correspondingly reduced, and more metabolic flows flow to the Leu synthetic pathway; the malonic acid can inhibit key enzymes of TCA cycle, so that the TCA cycle is weakened, the generation amount of acetic acid and other byproducts in the TCA cycle is reduced, more carbon sources enter a Leu synthesis way, and the output of Leu is improved; in the middle and later stages of fermentation, a certain amount of chitosan is added, amino on the chitosan is combined with teichoic acid or lipopolysaccharide with negative charges in bacterial cell walls, and cations such as Mg2+, Ca2+ and the like are chelated, so that the permeability of the cell walls is changed, and leucine is promoted to be secreted out of cells, and the yield of the leucine is improved; however, when the chitosan concentration is too high, a certain damage is caused to the strain, the strain proliferation is hindered, and death occurs.
The method adopts a multistage membrane coupling system to carry out separation and purification treatment on the L-leucine fermentation liquor, and can greatly remove mycoprotein and pigment in the fermentation liquor and greatly improve the product yield and the product purity through the multistage membrane graded continuous treatment; the invention adopts a multi-stage film coupling system, and has the advantages of high production continuity, high automation degree, quick and simple amplification and high production efficiency; in the three-stage extraction treatment of the multi-stage membrane coupling system, alcohol is used as an extractant, and the alcohol can be recycled after being used, so that the defects of high acid and alkali consumption and serious pollution in the traditional ion exchange process are overcome; in the treatment of the fermentation waste liquid, the fermentation waste liquid can be prepared into the mycoprotein feed and the organic-inorganic compound fertilizer, no pollutant is discharged in the whole process, and the method is green and environment-friendly.
Drawings
FIG. 1: influence of sodium acetate concentration on L-Leu yield;
FIG. 2: influence of sodium acetate concentration on Val and Hac content;
FIG. 3: the effect of malonic acid concentration on L-Leu production;
FIG. 4: effect of chitosan concentration on L-Leu production.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be described more clearly and completely below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A process for preparing and extracting L-leucine, comprising the steps of:
test strains: brevibacterium flavum: (Brevibacterium flavum）TK0303（Met-+IleL+2-TAr+α-ABr+β-HLr+Rifr+SGr) The institute of Metabolic engineering, university of Tianjin science and technology.
Obtaining Brevibacterium flavum seed liquid by conventional culture method, inoculating Brevibacterium flavum seed liquid into 500L fermentation tank containing 300L fermentation tank culture medium according to inoculation amount of 10%, inoculating concentration OD600Is 1.5; stirring at a rotation speed of 400 r/min; controlling the dissolved oxygen at 25% by stirring and ventilating; controlling the pH value to be 7.0 by automatically feeding ammonia water; the culture temperature is 33 ℃; defoaming with foam killer; when the fermentation time is 20 hours, feeding nutrient solution into the fermentation tank, maintaining the sugar concentration in the fermentation tank to be 1g/L, and stopping feeding 6 hours before the fermentation is finished; the fermentation period is 44 h.
Composition of fermenter Medium: glucose 50g/L, (NH)4)2SO410g/L of corn steep liquor, 10g/L of KH2PO4 4g/L，MgSO4·7H2O1.5 g/L, citric acid 0.5g/L, MnSO4·H2O 10mg/L，FeSO4·7H2O 0.1g/L，VB15mg/L, biotin 50. mu.g/L.
The nutrient solution comprises the following components: 100g/L of glucose, 5g/L of glycerol and 1g/L of betaine.
And when the fermentation time is 30 hours, adding a sodium acetate aqueous solution into the fermentation tank, and controlling the concentration of sodium acetate in the fermentation liquor to be 0.5g/L until the fermentation is finished, wherein the concentration of the sodium acetate aqueous solution is 100 g/L.
When the fermentation time is 30 hours, adding a malonic acid aqueous solution into the fermentation tank, and controlling the concentration of malonic acid in the fermentation liquid to be 0.2ml/L until the fermentation is finished; the concentration of the aqueous malonic acid solution was 15% (v/v).
When the fermentation time is up to 40h, adding chitosan into the fermentation tank, and controlling the concentration of the chitosan in the fermentation liquor to be 40 mg/L.
Taking leucine fermentation liquor, centrifuging for 5min at 4000rpm by adopting a high-speed disc centrifuge, and collecting filtrate and mycoprotein precipitation;
performing primary filtration treatment on the fermentation liquor by using the filtrate in a ceramic membrane of a multistage membrane coupling system, setting the filtration parameter pressure difference of 0.07MPa, the temperature of 50 ℃ and the pH value of 6.0, and collecting the filtered clear liquid;
carrying out active carbon decolorization treatment on the filtered clear liquid by a plate type closed filter, wherein the decolorization time is 65 min;
performing secondary decolorization treatment on the decolorized clear liquid in a decolorization membrane of a multistage membrane coupling system, setting decolorization process parameters of membrane inlet pressure of 0.9MPa, membrane outlet pressure of 0.8MPa and feed liquid flow rate of 120mL/min to obtain decolorized clear liquid;
evaporating and concentrating the decolorized clear liquid, and performing three-stage extraction treatment in sequential simulated moving bed chromatography of a multistage membrane coupling system, wherein the extraction process parameters are set to pH5.0, the upper column flow rate is 0.7BV/h, and the elution rate is 0.7BV/h, so as to obtain the extracted clear liquid;
and (4) carrying out four-effect concentration, centrifugation, fluidized bed drying and screen packaging on the extracted clear liquid to obtain the high-purity L-leucine. The detection calculation shows that the protein removal rate of the L-leucine fermentation liquor is 97.8%, the pigment removal rate is 98.3%, the product yield is 83.7%, and the product purity is 98.7%.
Example 2
A process for preparing and extracting L-leucine, comprising the steps of:
test strains: brevibacterium flavum: (Brevibacterium flavum）TK0303（Met-+IleL+2-TAr+α-ABr+β-HLr+Rifr+SGr) The institute of Metabolic engineering, university of Tianjin science and technology.
Obtaining Brevibacterium flavum seed liquid by conventional culture method, inoculating Brevibacterium flavum seed liquid into 500L fermentation tank filled with 300L fermentation tank culture medium according to 8% inoculation amount, inoculating concentration OD600Is 1.8; stirring speed is 350 r/min; controlling the dissolved oxygen at 25% by stirring and ventilating; controlling the pH value to be 6.8 by automatically feeding ammonia water; the culture temperature is 33 ℃; defoaming with foam killer; when the fermentation time is 20 hours, feeding nutrient solution into the fermentation tank, maintaining the sugar concentration in the fermentation tank to be 1g/L, and stopping feeding 6 hours before the fermentation is finished; the fermentation period is 44 h.
Composition of fermenter Medium: glucose 50g/L, (NH)4)2SO410g/L of corn steep liquor, 10g/L of KH2PO4 4g/L，MgSO4·7H2O1.5 g/L, citric acid 0.5g/L, MnSO4·H2O 10mg/L，FeSO4·7H2O 0.1g/L，VB15mg/L, biotin 50. mu.g/L.
The nutrient solution comprises the following components: 100g/L of glucose, 5g/L of glycerol and 1g/L of betaine.
And when the fermentation time is 30 hours, adding a sodium acetate aqueous solution into the fermentation tank, and controlling the concentration of sodium acetate in the fermentation liquor to be 0.4g/L until the fermentation is finished, wherein the concentration of the sodium acetate aqueous solution is 80 g/L.
When the fermentation time is 30 hours, adding a malonic acid aqueous solution into the fermentation tank, and controlling the concentration of malonic acid in the fermentation liquid to be 0.3ml/L until the fermentation is finished; the concentration of the aqueous malonic acid solution was 12% (v/v).
When the fermentation time is up to 40h, chitosan is added into the fermentation tank, and the concentration of the chitosan in the fermentation liquid is controlled to be 60 mg/L.
Taking leucine fermentation liquor, centrifuging for 4min at the speed of 5000rpm by adopting a high-speed disc centrifuge, and collecting filtrate and mycoprotein precipitation;
performing primary filtration treatment on the fermentation liquor by using the filtrate in a ceramic membrane of a multistage membrane coupling system, setting the filtration parameter pressure difference of 0.06MPa, the temperature of 50 ℃ and the pH value of 6.2, and collecting the filtered clear liquid;
performing active carbon decolorization treatment on the filtered clear liquid by using a plate type closed filter, wherein the decolorization time is 60 min;
performing secondary decolorization treatment on the decolorized clear liquid in a decolorization membrane of a multistage membrane coupling system, setting decolorization process parameters of membrane inlet pressure of 0.9MPa, membrane outlet pressure of 0.8MPa and feed liquid flow rate of 100mL/min to obtain decolorized clear liquid;
evaporating and concentrating the decolorized clear liquid, and performing three-stage extraction treatment in sequential simulated moving bed chromatography of a multistage membrane coupling system, wherein the extraction process parameters are set to pH5.0, the upper column flow rate is 0.6BV/h, and the elution rate is 0.6BV/h, so as to obtain the extracted clear liquid;
and (4) carrying out four-effect concentration, centrifugation, fluidized bed drying and screen packaging on the extracted clear liquid to obtain the high-purity L-leucine. The detection calculation shows that the protein removal rate of the L-leucine fermentation liquor is 97.4%, the pigment removal rate is 98.1%, the product yield is 83.4%, and the product purity is 98.6%.
Comparative example 1
Test strains: brevibacterium flavum: (Brevibacterium flavum）TK0303（Met-+IleL+2-TAr+α-ABr+β-HLr+Rifr+SGr) The institute of Metabolic engineering, university of Tianjin science and technology.
Obtaining Brevibacterium flavum seed liquid by conventional culture method, inoculating Brevibacterium flavum seed liquid into 500L fermentation tank containing 300L fermentation tank culture medium according to inoculation amount of 10%, inoculating concentration OD600Is 1.5; stirring at a rotation speed of 400 r/min; controlling the dissolved oxygen at 25% by stirring and ventilating; controlling the pH value to be 7.0 by automatically feeding ammonia water; the culture temperature is 33 ℃; defoaming with foam killer; when the fermentation time is 20 hours, feeding nutrient solution into the fermentation tank, maintaining the sugar concentration in the fermentation tank to be 1g/L, and stopping feeding 6 hours before the fermentation is finished; fermentation period is 44h to obtain fermentation liquor.
Example 3
1. On the basis of the conventional fermentation of comparative example 1, the sodium acetate is added into the fermentation liquor at a concentrationL-Leu and the main by-product content. The concentration gradients of sodium acetate in the fermentation liquor are set to be 0, 0.1,0.2,0.3,0.4,0.5,0.6 and 0.7 respectively, and the unit is g/L. As shown in FIGS. 1-2, the concentration of sodium acetate increased in the fermentation brothL-The content of Leu is steadily increased, the contents of the byproducts Val and Hac are gradually reduced, when the concentration of sodium acetate is increased to 0.5g/L, the content of L-Leu reaches the maximum value, the content of sodium acetate is continuously increased, the content of L-Leu is not obviously increased, at the moment, the contents of the byproducts Val and Hac are not continuously reduced, and the balance among metabolic flows is achieved. In the fermentation process, a proper amount of sodium acetate is added in the middle and later stages of fermentation, so that feedback inhibition can be performed on an acetic acid synthetic pathway, the acetic acid synthetic pathway is blocked, the generation amount of Val is correspondingly reduced, and more metabolic flows flow to a Leu synthetic pathway.
2. Selecting the concentration of sodium acetate in the fermentation liquor to be 0.5g/L, and verifying the addition concentration of malonic acid in the fermentation liquorL-Leu and the main by-product content. Setting different concentration gradients of malonic acid, 0, 0.1,0.2,0.3,0.4,0.5, unit ml/L, as shown in figure 3, by adding malonic acid in the middle and later stages of fermentation, and with the increase of concentration, the fermentation liquid isL-The content of Leu is increased, when the concentration of malonic acid is 0.3ml/L, the content of L-Leu is close to the peak value, the main reason is that the malonic acid can inhibit key enzymes of TCA cycle, further the TCA cycle is weakened, the generation amount of acetic acid and other byproducts in the TCA cycle is reduced, more carbon sources enter a Leu synthesis path, and therefore the yield of Leu is improved.
3. The concentration of sodium acetate in the fermentation liquor is 0.5g/L, the concentration of malonic acid is 0.3ml/L, and the chitosan pair is verifiedL-Leu content, generally speaking, a chitosan concentration of 100mg/L will inhibit the strain to some extent, so the chitosan concentration gradient is set to 0,5,10,20,40,60,80 in mg/L, and the fermentation is performed laterThe content of L-Leu in the fermentation liquid is high, secretion of L-Leu to the outside of cells is blocked, at the moment, the permeability of cell walls can be changed by adding chitosan, but when the concentration is too high, certain damage can be caused to the strain, the strain proliferation is blocked and the strain dies; the addition amount of 40-60mg/L is selected to be proper, and in this case, the content of L-Leu is high, but the concentration of the strain is not reduced.
The foregoing list is only illustrative of the preferred embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (10)
1. A process for preparing and extracting L-leucine, comprising the steps of:
step 1) fermentation, step 2) centrifugation, step 3) ceramic membrane filtration, step 4) activated carbon decolorization, step 5) decolorization membrane decolorization, step 6) sequential simulated moving bed chromatography extraction, and step 7) concentration, centrifugation and drying.
2. The process according to claim 1, characterized in that it comprises the following steps:
step 1) fermentation: inoculating the L-leucine-producing Brevibacterium flavum seed solution into a fermentation tank filled with a fermentation tank culture medium according to the inoculation amount of 8-10%, and stirring at the rotation speed of 300-; controlling the dissolved oxygen to be 20-30% by stirring and ventilating; controlling the pH value to be 7.0 by adding ammonia water in a flowing manner; the culture temperature is 33 ℃; defoaming with foam killer; when the fermentation time is 20 hours, feeding nutrient solution into the fermentation tank, maintaining the sugar concentration in the fermentation tank to be 1g/L, and stopping feeding 6 hours before the fermentation is finished; the fermentation period is 44 h;
step 2) centrifugation: taking the fermentation liquid, centrifuging for 3-5min at 4000-;
step 3) ceramic membrane filtration: performing primary filtration treatment on the fermentation liquor in the filter liquor obtained in the step 2) in a ceramic membrane of a multistage membrane coupling system, and collecting the filtered clear liquor;
step 4), activated carbon decolorization: performing active carbon decolorization treatment on the filtered clear liquid by using a plate-type closed filter, wherein the decolorization time is 60-65 min;
step 5) decoloring by a decoloring membrane: performing secondary decolorization treatment on the decolorized clear liquid in a decolorizing membrane of a multistage membrane coupling system to obtain decolorized clear liquid;
step 6) sequential simulated moving bed chromatographic extraction: evaporating and concentrating the decolorized clear liquid, and performing three-stage extraction treatment in a sequential simulated moving bed chromatography of a multistage membrane coupling system to obtain an extracted clear liquid;
step 7) concentrating, centrifuging and drying: and (4) carrying out four-effect concentration, centrifugation, fluidized bed drying and screen packaging on the extracted clear liquid to obtain the high-purity L-leucine.
3. The process according to claim 2,
when the fermentation is carried out for 30 hours, adding a sodium acetate aqueous solution into the fermentation tank, and controlling the concentration of sodium acetate in the fermentation liquor to be 0.1-1g/L until the fermentation is finished, wherein the concentration of the sodium acetate aqueous solution is 50-100 g/L;
when the fermentation time is 30 hours, adding a malonic acid aqueous solution into the fermentation tank, and controlling the concentration of malonic acid in the fermentation liquid to be 0.1-0.5 ml/L until the fermentation is finished; the concentration of the malonic acid aqueous solution is 10-20%;
when the fermentation time is up to 40h, adding chitosan into the fermentation tank, and controlling the concentration of the chitosan in the fermentation liquid to be 20-80mg/L until the fermentation is finished.
4. The process of claim 2, wherein the components of the fermentor medium are: glucose 50g/L, (NH)4)2SO410g/L of corn steep liquor, 10g/L of KH2PO4 4g/L，MgSO4·7H2O1.5 g/L, citric acid 0.5g/L, MnSO4·H2O 10mg/L，FeSO4·7H2O 0.1g/L，VB15mg/L, biotin 50. mu.g/L.
5. The process according to claim 2, characterized in that the components of the nutrient solution are: 100g/L of glucose, 5g/L of glycerol and 1g/L of betaine.
6. The process of claim 3, wherein the concentration of sodium acetate in the fermentation broth is controlled to be 0.4-0.5 g/L; controlling the concentration of malonic acid in the fermentation liquor to be 0.2-0.3 ml/L; the concentration of chitosan in the fermentation liquor is controlled to be 40-50 mg/L.
7. The process according to claim 2, wherein the ceramic membrane filtration parameters are: the pressure difference is 0.06-0.07MPa, the temperature is 50 ℃, and the pH value is 6.0-6.5.
8. The process according to claim 2, wherein the decolorization parameters of the decolorizing membrane are: the membrane inlet pressure is 0.9MPa, the membrane outlet pressure is 0.8MPa, and the flow rate of the feed liquid is 120 mL/min.
9. The process according to claim 2, wherein the parameters of the sequential simulated moving bed chromatography extraction are: pH5.0, flow rate of column feeding 0.7BV/h, and elution speed 0.7 BV/h.
10. L-leucine obtainable by a process according to claims 1-9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911003541.9A CN110607331B (en) | 2019-10-22 | 2019-10-22 | Process for preparing and extracting L-leucine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911003541.9A CN110607331B (en) | 2019-10-22 | 2019-10-22 | Process for preparing and extracting L-leucine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110607331A true CN110607331A (en) | 2019-12-24 |
| CN110607331B CN110607331B (en) | 2023-03-10 |
Family
ID=68894977
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911003541.9A Active CN110607331B (en) | 2019-10-22 | 2019-10-22 | Process for preparing and extracting L-leucine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110607331B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0167132A2 (en) * | 1984-06-29 | 1986-01-08 | Ajinomoto Co., Inc. | Process for producing l-isoleucine by fermentation |
| JPS6115695A (en) * | 1984-06-29 | 1986-01-23 | Ajinomoto Co Inc | Preparation of l-isoleucine by fermentation method |
| CN1140761A (en) * | 1995-03-30 | 1997-01-22 | 味之素株式会社 | Process for producing L-leucine |
| CN105274180A (en) * | 2015-10-28 | 2016-01-27 | 新疆阜丰生物科技有限公司 | Method for continuously extracting leucine by aid of chromatograms of sequential simulated moving beds |
| CN108084041A (en) * | 2017-12-20 | 2018-05-29 | 大连医诺生物股份有限公司 | A kind of method of efficient process zymotic fluid production amino acid |
| CN109609564A (en) * | 2018-12-30 | 2019-04-12 | 新疆阜丰生物科技有限公司 | A method of improving L-Leu fermentation yield |
| CN109761833A (en) * | 2019-02-24 | 2019-05-17 | 内蒙古拜克生物有限公司 | A kind of isolation and purification method of L-Leu |
-
2019
- 2019-10-22 CN CN201911003541.9A patent/CN110607331B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0167132A2 (en) * | 1984-06-29 | 1986-01-08 | Ajinomoto Co., Inc. | Process for producing l-isoleucine by fermentation |
| JPS6115695A (en) * | 1984-06-29 | 1986-01-23 | Ajinomoto Co Inc | Preparation of l-isoleucine by fermentation method |
| CN1140761A (en) * | 1995-03-30 | 1997-01-22 | 味之素株式会社 | Process for producing L-leucine |
| CN105274180A (en) * | 2015-10-28 | 2016-01-27 | 新疆阜丰生物科技有限公司 | Method for continuously extracting leucine by aid of chromatograms of sequential simulated moving beds |
| CN108084041A (en) * | 2017-12-20 | 2018-05-29 | 大连医诺生物股份有限公司 | A kind of method of efficient process zymotic fluid production amino acid |
| CN109609564A (en) * | 2018-12-30 | 2019-04-12 | 新疆阜丰生物科技有限公司 | A method of improving L-Leu fermentation yield |
| CN109761833A (en) * | 2019-02-24 | 2019-05-17 | 内蒙古拜克生物有限公司 | A kind of isolation and purification method of L-Leu |
Non-Patent Citations (2)
| Title |
|---|
| NANNINGA 等: "Amino acid fermentation and hydrogen transfer in mixed cultures", 《FEMS MICROBIOLOGY ECOLOOY》 * |
| 张玉富 等: "生物素及膜偶联间歇透析发酵对黄色短杆菌生产L-亮氨酸的影响", 《食品与发酵工业》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110607331B (en) | 2023-03-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109504719B (en) | Method for improving acid production rate and extraction rate of glutamic acid | |
| CN107058416B (en) | Fermentation process for refining glutamic acid | |
| WO2018228246A1 (en) | Method for enzymatic preparation of glutathione | |
| CN102559796B (en) | Method for preparing gardenia blue pigment by utilizing immobilized yeast cells | |
| CN103320362B (en) | One strain is produced the bacterial strain of L-Glutamic decarboxylase and is produced the method for γ-aminobutyric acid with it | |
| CN109504720B (en) | Green production process of glutamic acid | |
| CN109486894A (en) | A kind of production method of Valine | |
| CN104745666A (en) | New technology for extracting L-glutamine | |
| CN107099563B (en) | Method for preparing monosodium glutamate by using isoelectric technology | |
| CN101781346A (en) | Method for separating uridylic acid from biocatalytic conversion solution | |
| CN116200433A (en) | Method for preparing ergothioneine by biosynthesis of Cordyceps fungus | |
| CN113621658B (en) | Preparation method for continuously producing 1,3-dihydroxyacetone and erythrulose | |
| CN105274179A (en) | Process for extracting L-isoleucine | |
| CN111004822A (en) | Production process of high-purity threonine | |
| CN111057727B (en) | Method for producing, separating and extracting L-glutamine | |
| CN113321580B (en) | Method for producing malic acid | |
| CN104651419A (en) | Method for coproducing mannitol and D-lactic acid by microbial anaerobic fermentation | |
| CN109136299B (en) | Method for preparing, extracting and purifying threonine | |
| CN117126898A (en) | Process for preparing valine by biotechnology | |
| CN111139273B (en) | Method for preparing, separating and extracting L-tryptophan | |
| CN110607331B (en) | Process for preparing and extracting L-leucine | |
| CN103361394A (en) | Method for preparing 9alpha-hydroxide-androstenedione by utilizing microbial conversion | |
| CN110592154B (en) | Process for producing and extracting tryptophan | |
| CN110541013B (en) | Method for producing L-leucine by fermentation | |
| CN112481330B (en) | Algae source beta-1, 3-glucan fermentation production method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200217 Address after: 831302 Xinjiang Fufeng Biotechnology Co., Ltd. ganquanbao Industrial Zone, Urumqi Economic and Technological Development Zone (Toutunhe District), Xinjiang Uygur Autonomous Region Applicant after: XINJIANG FUFENG BIOTECHNOLOGY Co.,Ltd. Address before: 830026 Xinjiang Fufeng Biotechnology Co., Ltd. ganquanbao Industrial Zone, Urumqi Economic and Technological Development Zone (Toutunhe District), Xinjiang Uygur Autonomous Region Applicant before: Feng Shihong |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |