CN109486876A - A method of threonine is extracted and is purified in fermentation - Google Patents
A method of threonine is extracted and is purified in fermentation Download PDFInfo
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Abstract
The invention belongs to threonine production technical fields, disclose a kind of method for fermenting, extracting and purifying threonine, it includes the following steps: that step 1) is fermented, step 2 separation of fermentative broth, step 3) condensing crystallizing, step 4) separate crystal, step 5) is dry, mother liquor crystallization of step 6) and separation, step 7) chromatographic isolation, step 8) crystallization and separation.Comprehensive utilization of the extraction and purification process of the present invention to threonine fermentation liquid and waste liquor, not only increases threonine yield and purity, brings the benefit in production, also reduces environmental protection pressure.
Description
Technical field
The invention belongs to threonine production technical fields, and in particular to a method of threonine is extracted and is purified in fermentation.
Background technique
L-threonine (hereinafter referred to as threonine) is white crystals or crystalline powder, is a kind of polar amino acid, odorless, taste
It is slightly sweet.253 DEG C melt and decompose.Water is dissolved under high temperature, 25 DEG C of solubility are 20.5g/100ml, do not dissolve in ethyl alcohol, ether and chlorine
It is imitative.Specific rotatory power is 27.6 ° -29 °, iso-electric point 5.64.
External threonine industry development is more early, the U.S., France, Germany, Japan and other countries to the synthesis of threonine, separation,
Using etc. carried out a series of research, especially Japan fermentation method produce threonine in terms of have certain advantage, taste
Element company be maximum threonine manufacturing enterprise in the world, the threonine of the said firm's production occupies 60% or more world market
Share, aginomoto European Company threonine productivity are 3.5 ten thousand tons, 10,000 tons of production capacity of aginomoto Heetleand company,
The said firm is to expand occupation rate of market, its threonine production capacity is increased to 50,000 tons by plan.German goldschmidt chemical corporation is also threonine
Big producer, and be the enterprise that the world uniquely can be produced at the same time three big amino acid products (egg relies, Soviet Union), it is at this at present
Factory's threonine production capacity that Lip river is cut down gram at 10,000 tons plans that threonine production capacity is promoted to ten thousand tons of 2-3 within the coming years.Day
This consonance Kyowa Hakko and ADM company, the U.S. possess 10,000 tons and 5000 tons of threonine production scale respectively, furthermore South Korea
Khidze Co., Ltd, company, Cheil Samsung Indonesia etc. also produce threonine.
China's threonine industry development is later, domestic before the nineties in last century not produce substantially, the nineties with
Just start to produce afterwards.By development in 10 years, also fail to break through 200 tons to yield at the end of the 20th century, the staple market of product is also
Pharmaceuticals industry, be mainly used for various amino acid transfusions (amino acid transfusion is usually used in physical recovery perioperatively, it is malnutritive and
The adjuvant treatment of chronic wasting disease etc. is the very big kind of quantity.In recent years, the big portion of domestic production producer threonine
Point supply domestic demand, and the oriented state's outer vent amino acid of part company, no matter the production of threonine is from technical indicator, cost
From index, product quality, domestic production is horizontal still with foreign countries there are certain gap, and product price totally compares external price
It is slightly higher.Nearly 2 years, with increasing for manufacturer, threonine produced the growth that increasingly tends to be steady, and had been gradually reduced and other factory, state
The gap of family.Along with the extensive use of international market threonine, domestic some well-known Feed Enterprises also start largely raising
Threonine is applied in material, to improve the quality of feed, the needs of further satisfaction feed industry.
Currently, the production method of threonine mainly has fermentation method, protein Hydrolyze method and 3 kinds of chemical synthesis, microorganism
Fermentation method produces threonine, because the advantages that its simple process and low cost has become current main stream approach.L-threonine it is main
Production bacterial strain has brevibacterium flavum and Escherichia coli.Threonine Fermentation technology is primarily present that fermentation efficiency is low and purity at present
Defect not up to standard.Bacterial strain is transformed all the time by various methods to improve the yield of amino acid in people, and early stage is most widely used
Be it is various under the conditions of mutation breeding, it is purposive right on a molecular scale and with the exposition of amino acid bio metabolic pathway
Metabolic pathway is transformed also appears its advantage gradually, in addition, the fermentation condition optimization process in middle reaches and the recycling in downstream are net
Change process is also an emphasis.
Colibacillus engineering strain is the main bacterial strain of microbe industrial fermentation production threonine, generates L- Soviet Union in fermentation
While propylhomoserin, the metabolic by-products such as acetic acid, alanine, valine and arginine can be also generated, influence bacterium to a certain extent
Body growth and the synthesis and accumulation of L-threonine.Wherein, the inhibitory effect of acetic acid is particularly evident, when Acetic Acid Accumulation is to certain dense
When spending, the specific growth rate of thallus declines rapidly, and Product formation substantially reduces, and forms vicious circle, while foreign gene
Expression is also heavily affected.Acetic Acid Accumulation is adversely affected caused by cell metabolism, is using Escherichia coli as host strain
Express a very important problem of foreign protein.How the content of acetic acid is reduced, to improve biomass and threonine
Yield is the emphasis that we study.Document " control of acetic acid in L-threonine fermentation process, science and technology of fermenting communication 2012 " exists
During Escherichia coli fermentation prepares threonine, the generation of its by-product acetic acid is controlled by selecting suitable fermentation condition,
It can reduce the generation of acetic acid, but reduce amplitude and be reduced to unobvious, the yield of threonine cannot be greatly improved, there is no industry
Change the value promoted.The culture medium that patented technology " a kind of ultralow moisture content threonine production method " before applicant is recorded: Portugal
Grape sugar 80g/L, corn pulp 20g/L, ammonium sulfate 2g/L, calcium carbonate 0.75g/L, KH2PO40.2g/L, K2HPO4 0.2g/L, NaCl
0.2g/L, pH value 6.5；The content of threonine can reach 10g/100ml in fermentation liquid, but the phase is dead after fermentation for bacterial strain
Rate is higher, and glucose utilization is larger, also there is to be hoisted threonine yield in fermentation liquid.Patented technology before applicant
" a kind of method of preparing granular type threonine " improves the yield of threonine by mixed fermentation technology, but extraction process
In, mother liquor is not extracted, causes waste.
Summary of the invention
On the basis of research achievement before, the invention proposes a kind of fermentations, the method extracted and purify threonine.It should
Method develops the production decision of low-carbon environment-friendly, gives full play to the advantage in resources, and waste liquor is effectively utilized, and fills it
The resource-effective utilization divided.
The present invention is achieved by the following technical solution:
A method of threonine is extracted and is purified in fermentation comprising following steps: step 1) fermentation, step 2 fermentation liquid point
From step 3) condensing crystallizing, step 4) separates crystal, and step 5) is dry, mother liquor crystallization of step 6) and separation, step 7) color
Spectrum separation, step 8) crystallization and separation.
Further, the step 1) fermentation, comprising: by colibacillus engineering seed liquor according to the inoculum concentration of 5-10%
It is linked into the fermentor containing fermentation medium and ferments, then fermentation time 36h connects according to the inoculum concentration of 5-10%
Enter Chlamydomonas reinhardtii, continue fermented and cultured 48h, stop fermentation, collects fermentation liquid.
Preferably, the component of the fermentation medium are as follows: glucose 20g/L, glycerol 20g/L, corn pulp 20g/L, sulfuric acid
Ammonium 2g/L, potassium dihydrogen phosphate 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, epsom salt 0.1g/L, ferrous sulfate heptahydrate 0.01g/
L, manganese sulfate monohydrate 0.01g/L, pH value 6.5.
Further, the step 2 separation of fermentative broth, comprising: fermentation liquid is centrifuged by disk plate centrifuge, collects upper layer
Liquid and precipitating；Supernatant liquid passes through ceramic membrane filter, collects filtered solution, filtered solution is separated through decanter centrifuge, centrifugation speed
Degree is 5000rpm, centrifugation time 3min, collects supernatant；Then pass through ultrafiltration membrance filter, collect permeate.
Further, the step 3) condensing crystallizing, comprising: will transmit through liquid and crystallized with intermittent single-action condensing crystallizing pot.
Further, the step 4) separates crystal: being centrifuged with centrifuge, collects crystal and a mother liquor.
Further, the step 5) is dry, comprising: the crystal isolated is dried, and obtains threonine product.
Further, mother liquor crystallization of the step 6) and separation, comprising: a mother liquor is subjected to condensing crystallizing, so
After be centrifuged, collect crystal and secondary mother liquid.
Further, the step 7) chromatographic isolation, comprising: secondary mother liquid utilizes sequential simulated moving bed carry out chromatography
Separation obtains the separating liquid rich in threonine；
Further, the step 8) crystallization and separation, comprising: separating liquid obtained by step 7) is subjected to crystallization treatment, centrifugation point
From crystal, then with step 6) obtained by crystal merge, return step 3) in intermittent single-action condensing crystallizing pot in.
The beneficial effect of starting point and acquirement that the present invention studies mainly includes but is not limited to the following aspects:
Fermenting carbon source selection glucose and glycerol of the present invention, earlier fermentation, cell density is low, and oxygen-supplying amount is sufficient, and Escherichia coli are excellent
First with glucose as carbon source, the generation of growing microorganism and threonine can be promoted；It fermenting the middle and later periods, glucose is depleted,
Escherichia coli use glycerol as carbon source at this time, and since the rate that cell absorbs glycerol is lower, the carbon flow into glycolysis declines,
To reduce the accumulation of acetic acid, while improving the yield of threonine；
The present invention can carry out non-light and work as carbon source using the acetic acid in fermentation liquid by being inoculated with Chlamydomonas reinhardtii in fermentation
With, and it is more difficult use glycerol as carbon source, thus relieve to Escherichia coli produce threonine inhibiting effect, additionally it is possible to carry out micro-
The photosynthesis of amount discharges oxygen, for Escherichia coli fermentation produce threonine come using.By adding Chlamydomonas reinhardtii, it is not only able to mention
The yield of high threonine, and mycoprotein yield also correspondinglys increase.
Comprehensive utilization of the extraction and purification process of the present invention to threonine fermentation liquid and waste liquor not only increases threonine receipts
Rate also reduces environmental protection pressure, brings the benefit in production.
Figure of description
Fig. 1: the yield of acetic acid of each group in different time points.
Specific embodiment
Those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that
All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair
It is bright.Product and method of the invention is described by preferred embodiment, and related personnel can obviously not depart from this hair
Product as described herein and method are modified in bright content, spirit and scope or appropriate changes and combinations, to realize and answer
Use the technology of the present invention.For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.
Embodiment 1
A method of threonine is extracted and is purified in fermentation comprising following steps:
Step 1) fermentation: by colibacillus engineering K12 △ dapA seed liquor, (concentration of seed liquor is 1 × 108Cfu/mL it) presses
It is linked into the fermentor containing fermentation medium and ferments according to 10% inoculum concentration, 30 DEG C of temperature, tank pressure is 0.04MPa, is led to
Tolerance is 0.5vvm, revolving speed 100rpm, fermentation time 36h, then accesses Chlamydomonas reinhardtii (Rhein according to 10% inoculum concentration
The concentration of chlamydomonas is 1 × 105Cfu/mL), continue fermented and cultured 48h, stop fermentation, collect fermentation liquid；
The component of the fermentation medium are as follows: glucose 20g/L, glycerol 20g/L, corn pulp 20g/L, ammonium sulfate 2g/L, phosphoric acid
Potassium dihydrogen 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, epsom salt 0.1g/L, ferrous sulfate heptahydrate 0.01g/L, sulfuric acid monohydrate
Manganese 0.01g/L, pH value 6.5；
Step 2 separation of fermentative broth: fermentation liquid first pass around disk plate centrifuge with 4000rpm be centrifuged 5min, collect supernatant liquid and
Precipitating；Supernatant liquid is filtered by ceramic membrane (10,000 Da molecular cut off), filtered solution is collected, by filtered solution through decanter centrifuge
Separation, centrifugal speed 5000rpm, centrifugation time 3min collect supernatant；Then pass through ultrafiltration membrance filter, collect and penetrate
Liquid, ultrafiltration retaining molecular weight are 300Da；
Step 3) condensing crystallizing: it will transmit through liquid by evaporator and be concentrated into the 1/3 of stoste volume, with intermittent single-action condensing crystallizing
Pot crystallization, starts when feed liquid is concentrated into Baume degrees 27-30 in pot plus crystal seed, crystallization process control 65 DEG C of temperature or so, vacuum
Spend 0.08MPa or so；When crystal size is between 0.7-1.8mm in pot；
Step 4) separates crystal: being centrifuged with centrifuge, centrifuge speed is risen to by 400-500r/min with the rate of 10r/s
Then 900-980r/min maintains revolving speed centrifugation 150s of 900-980r/min or so, finally collects crystal and a mother liquor；
Step 5) is dry: the crystal isolated is dried, and obtains threonine product；
Mother liquor crystallization of step 6) and separation: a mother liquor is subjected to condensing crystallizing, is then centrifuged for, collects crystal and secondary mother
Liquid；
Step 7) chromatographic isolation: secondary mother liquid utilizes sequential simulated moving bed carry out chromatographic isolation, removal sugar, salt and pigment
Equal impurity, obtain the separating liquid rich in threonine；
Step 8) crystallization and separation: carrying out crystallization treatment for separating liquid obtained by step 7), be centrifugated crystal, then with step 6)
Gained crystal merges, and squeezes into the intermittent single-action condensing crystallizing pot in step 3), is crystallized.
Embodiment 2
A method of threonine is extracted and is purified in fermentation comprising following steps:
Step 1) fermentation: by colibacillus engineering K12 △ dapA seed liquor, (concentration of seed liquor is 1 × 108Cfu/mL it) presses
It is linked into the fermentor containing fermentation medium and ferments according to 6% inoculum concentration, 32 DEG C of temperature, tank pressure is 0.04MPa, is led to
Tolerance is 0.4vvm, and revolving speed 100rpm, fermentation time culture is 48h, then accesses Chlamydomonas reinhardtii (Lay according to 8% inoculum concentration
The concentration of mattress chlamydomonas is 1 × 105Cfu/mL), continue fermented and cultured 48h, stop fermentation, collect fermentation liquid；
The component of the fermentation medium are as follows: glucose 30g/L, glycerol 30g/L, corn pulp 30g/L, ammonium sulfate 3g/L, phosphoric acid
Potassium dihydrogen 0.3g/L, dipotassium hydrogen phosphate 0.3g/L, epsom salt 0.2g/L, ferrous sulfate heptahydrate 0.02g/L, manganese sulfate monohydrate
0.02g/L, pH value 6.8；
Step 2 separation of fermentative broth: fermentation liquid first pass around disk plate centrifuge with 4000rpm be centrifuged 5min, collect supernatant liquid and
Precipitating；Supernatant liquid is filtered by ceramic membrane (10,000 Da molecular cut off), filtered solution is collected, by filtered solution through decanter centrifuge
Separation, centrifugal speed 5000rpm, centrifugation time 3min collect supernatant；Then pass through ultrafiltration membrance filter, collect and penetrate
Liquid, ultrafiltration retaining molecular weight are 300Da；
Step 3) condensing crystallizing: it will transmit through liquid by evaporator and be concentrated into the 1/3 of stoste volume, with intermittent single-action condensing crystallizing
Pot crystallization, starts when feed liquid is concentrated into Baume degrees 27-30 in pot plus crystal seed, crystallization process control 65 DEG C of temperature or so, vacuum
Spend 0.08MPa or so；When crystal size is between 0.7-1.8mm in pot；
Step 4) separates crystal: being centrifuged with centrifuge, centrifuge speed is risen to by 400-500r/min with the rate of 10r/s
Then 900-980r/min maintains revolving speed centrifugation 150s of 900-980r/min or so, finally collects crystal and a mother liquor；
Step 5) is dry: the crystal isolated is dried, and obtains threonine product；
Mother liquor crystallization of step 6) and separation: a mother liquor is subjected to condensing crystallizing, is then centrifuged for, collects crystal and secondary mother
Liquid；
Step 7) chromatographic isolation: secondary mother liquid utilizes sequential simulated moving bed carry out chromatographic isolation, removal sugar, salt and pigment
Equal impurity, obtain the separating liquid rich in threonine；
Step 8) crystallization and separation: carrying out crystallization treatment for separating liquid obtained by step 7), be centrifugated crystal, then with step 6)
Gained crystal merges, and squeezes into the intermittent single-action condensing crystallizing pot in step 3), is crystallized.
Embodiment 3
Influence of the different factors to production amount of threonine and yield of acetic acid in present invention process:
Group is set:
Experimental group: embodiment 1；
Control group 1: not adding Chlamydomonas reinhardtii, remaining is the same as embodiment 1；
Glycerol: being replaced with the glucose of equal quality by control group 2, remaining is the same as embodiment 1；
Control group 3: not adding Chlamydomonas reinhardtii, while glycerol being replaced with to the glucose of equal quality, remaining is the same as embodiment 1.
The content of threonine and acetic acid is shown in Table 1 in each final fermentation liquid of group:
Table 1
| Group | Threonine g/L | Acetic acid g/L |
| Experimental group | 137.1 | 0.6 |
| Control group 1 | 102.5 | 13.9 |
| Control group 2 | 118.2 | 4.7 |
| Control group 3 | 96.4 | 15.6 |
Conclusion: experimental group can utilize the acetic acid in threonine fermentation liquid by carrying out assisted fermentation processing to Chlamydomonas reinhardtii
Non- light and effect are carried out as carbon source, to relieve the inhibiting effect that Escherichia coli are produced with threonine, additionally it is possible to carry out micro
Photosynthesis discharge oxygen, for Escherichia coli fermentation produce threonine come using；Part glucose is substituted by glycerol simultaneously, with
The consumption of glucose, Escherichia coli use glycerol as carbon source, due to cell absorb glycerol rate it is lower, reduce acetic acid
Accumulation, while improving the yield of threonine, pass through each group comparative test and find, compared with control group 1-3, the present invention
The production amount of threonine of experimental group is respectively increased 33.76%, 15.99%, 42.22%；And experimental group acetic acid content is only 0.6g/L, phase
When in the 3.85% of control group 1.
The present invention also has detected the yield of acetic acid of each group in different time points, by taking embodiment 1 as an example, chooses fermentation respectively
Afterwards, 36h, 48h, 60h, 72h, 84h, totally 5 time points are detected, and concrete outcome is shown in Fig. 1.In experimental group,
With the increase of fermentation time, acetic acid content is reduced rapidly, and is relieved the synthesis to threonine and is inhibited, to improve threonine
Secretory volume；And in control group 1 and 3, due to not adding Chlamydomonas reinhardtii, acetic acid content is caused to continue growing；Control group 2
In, due to being added to Chlamydomonas reinhardtii, so that acetic acid content gradually declines, but fall is lower than experimental group, it may be possible to because
Experimental group is added to glycerol as carbon source, and the carbon source into acetate pathway reduces, and Chlamydomonas reinhardtii is more difficult uses glycerol as
Carbon source is only capable of using acetic acid, and then causes the fall of experimental group acetic acid more obvious.
Embodiment 4
The present invention adopt new technology process production threonine stable product quality, main indicator be substantially better than " feed grade L- Soviet Union
Propylhomoserin " (GB/T21979-2008) requirement.Through third-party testing product performance index such as the following table 2:
Table 2
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, although this
Invention has been described by way of example and in terms of the preferred embodiments, however, be not intended to limit the invention, any person skilled in the art,
Without departing from the scope of the present invention, a little change or modification can be made using the technology contents disclosed certainly, become
The equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, it is right according to the technical essence of the invention
Any simple modification, equivalent change and modification made by above embodiments, belong in the range of technical solution of the present invention.
Claims (10)
1. a kind of fermentation, the method extracted and purify threonine comprising following steps: step 1) fermentation, step 2 fermentation liquid
Separation, step 3) condensing crystallizing, step 4) separate crystal, and step 5) is dry, mother liquor crystallization of step 6) and separation, step 7)
Chromatographic isolation, step 8) crystallization and separation.
2. the method according to claim 1, wherein the step 1) is fermented, comprising: by colibacillus engineering
Seed liquor is linked into the fermentor containing fermentation medium according to the inoculum concentration of 5-10% ferments, fermentation time 36h,
Then Chlamydomonas reinhardtii is accessed according to the inoculum concentration of 5-10%, continues fermented and cultured 48h, stop fermentation, collect fermentation liquid.
3. according to the method described in claim 2, it is characterized in that, the component of the fermentation medium are as follows: glucose 20g/L,
Glycerol 20g/L, corn pulp 20g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, epsom salt
0.1g/L, ferrous sulfate heptahydrate 0.01g/L, manganese sulfate monohydrate 0.01g/L, pH value 6.5.
4. according to the method described in claim 2, it is characterized in that, the step 2 separation of fermentative broth, comprising: fermentation liquid passes through
Disk plate centrifuge centrifugation, collects supernatant liquid and precipitating；Supernatant liquid passes through ceramic membrane filter, filtered solution is collected, by filtered solution
It is separated through decanter centrifuge, centrifugal speed 5000rpm, centrifugation time 3min, collects supernatant；Then pass through ultrafiltration membrane mistake
Permeate is collected in filter.
5. according to the method described in claim 4, it is characterized in that, the step 3) condensing crystallizing, comprising: will transmit through liquid use between
The crystallization of formula of having a rest single-action condensing crystallizing pot.
6. according to the method described in claim 5, it is characterized in that, the step 4) separates crystal: it is centrifuged with centrifuge,
Collect crystal and a mother liquor.
7. according to the method described in claim 6, it is characterized in that, the step 5) is dry, comprising: crystal is dried,
Obtain threonine product.
8. method according to claim 6 or 7, which is characterized in that mother liquor crystallization of the step 6) and separation, packet
It includes: a mother liquor being subjected to condensing crystallizing, is then centrifuged for, collects crystal and secondary mother liquid.
9. according to the method described in claim 8, it is characterized in that, the step 7) chromatographic isolation, comprising: secondary mother liquid utilizes
Sequential simulated moving bed carry out chromatographic isolation obtains the separating liquid rich in threonine.
10. according to the method described in claim 9, it is characterized in that, step 8) crystallization and separation, comprising: by step 7)
Gained separating liquid carry out crystallization treatment, be centrifugated crystal, then with step 6) obtained by crystal merge, return step 3) between
In formula of having a rest single-action condensing crystallizing pot.
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| CN110846348A (en) * | 2019-08-29 | 2020-02-28 | 赵兰坤 | Preparation method of threonine fermentation medium |
| CN110846350A (en) * | 2019-09-12 | 2020-02-28 | 赵兰坤 | Threonine production and separation refining process |
| CN110846351A (en) * | 2019-12-22 | 2020-02-28 | 赵兰坤 | Threonine fermentation medium prepared by using mycoprotein as raw material |
| CN112552198A (en) * | 2020-12-30 | 2021-03-26 | 南通紫琅生物医药科技有限公司 | Separation and extraction method of amino acid mother liquor |
| CN113336662A (en) * | 2021-04-18 | 2021-09-03 | 呼伦贝尔东北阜丰生物科技有限公司 | Process for improving extraction crystallization of threonine |
| CN113774096A (en) * | 2021-10-21 | 2021-12-10 | 呼伦贝尔东北阜丰生物科技有限公司 | Optimization method of threonine production and extraction process |
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| CN110846348A (en) * | 2019-08-29 | 2020-02-28 | 赵兰坤 | Preparation method of threonine fermentation medium |
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| CN112552198A (en) * | 2020-12-30 | 2021-03-26 | 南通紫琅生物医药科技有限公司 | Separation and extraction method of amino acid mother liquor |
| CN113336662A (en) * | 2021-04-18 | 2021-09-03 | 呼伦贝尔东北阜丰生物科技有限公司 | Process for improving extraction crystallization of threonine |
| CN113774096A (en) * | 2021-10-21 | 2021-12-10 | 呼伦贝尔东北阜丰生物科技有限公司 | Optimization method of threonine production and extraction process |
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