CN103525881A - Method for producing L-aspartic acid converted from fumaric acid through Escherichia coli undergoing fermentation culture in large pot with volume of 20m<3> - Google Patents
Method for producing L-aspartic acid converted from fumaric acid through Escherichia coli undergoing fermentation culture in large pot with volume of 20m<3> Download PDFInfo
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Abstract
The invention relates to a method for producing L-aspartic acid converted from fumaric acid through Escherichia coli undergoing fermentation culture in a large pot with a volume of 20m<3>. A fluid medium 90% (v/v) is added in a large-scale fermentation pot, the diameter D of the pot body is 2200mm, and the height H0 of the pot barrel part is 5280mm. An Escherichia coli seed solution is inoculated, and the inoculation amount is 0.012% (v/v). The above mixture is cultured for 13h at a ventilation quantity of 280m<3>/h, at a rotating speed of 100rpm and at a pot pressure of 0.05MPa. A fermentation solution with a viable organism quantity of 1.89*108cfu/ml and an L-aspartic acid enzyme activity of 4725U/ml is prepared. Fumaric acid is converted through the fermentation solution and L-aspartic acid is obtained. The key of the method is that in the 20m<3> large pot fermentation of Escherichia coli HY-05C, the inoculation amount is low, only 2.1L of the shake flask seed solution is inoculated directly, the fermentation culture time is 13h, and a fermentation solution with high L-aspartic acid enzyme activity is obtained. Step by step amplification culture of seed pots is saved, and risks of bacterial infection during the seed amplification culture process are avoided. The production cycle is shortened and the production efficiency is raised.
Description
Technical field
The present invention relates to microbial fermentation technology field, especially relate to a kind of method that industry application bulk fermentation is produced L-Aspartic acid.
Background technology
L-Aspartic acid is a kind of important amino acid, is the main raw material of production aspartame, poly aspartic acid, ALANINE.And the market requirement of these products is also strengthening year by year.
Aspartame is a kind of novel sweeting agent, and sugariness is about 200 times of sucrose, is applicable to cardiovascular diseases and obesity crowd consumption.The whole world every year need be by approximately 60000 tons of aspartame, and demand is huge, for L-Aspartic acid provides the great market space.Poly aspartic acid is a kind of environmental protection macromolecular material, its nontoxic, readily biodegradable, have good wetting ability and water-soluble, as inhibiter, sequestrant and the dispersion agent of high-quality, be widely used in water treatment, fertilizer, washing, oil production, metal and cut and cut and the fields such as makeup.Poly aspartic acid is a kind rising tendency of turning over every year in the demand of every field.ALANINE is a kind of foodstuff additive, is also the important source material of producing vitamin B6 and aminopropanol.
L-Aspartic acid product has the huge market space, approximately 300,000 tons of global annual requirements.China has overwhelming superiority in this products production field at present, and how further promoting production level will contribute to promote the fast and stable development of China's L-Aspartic acid and related industries thereof.
Colon bacillus has certain application foundation in L-Aspartic acid biotransformation method is produced.Meanwhile, colon bacillus is the type strain of protokaryon, and genetic background is clear, studies very detailed.Colon bacillus and its engineering bacteria are widely used in production classes of compounds.Colon bacillus is applied to ASPARTIC ACID and has following advantage: the pathways metabolism of (1) colon bacillus is clear, and genetic modification method is various, can carry out easily metabolic engineering; (2) existing more colon bacillus or its engineering bacteria are produced the research of various product, produce other products experience is provided for colon bacillus; (3) fermentation of colon bacillus high-density or large system also has more research, and the production that its corresponding processing parameter and equipment flowsheet are ASPARTIC ACID provides foundation.
The sixties in 20th century, a day Honda limit drugmaker utilizes FUMARIC ACID TECH GRADE fermentation technique to take the lead in realizing the suitability for industrialized production of L-Aspartic acid.Along with the development of immobilization technology, 1973 Nian Tian limit drugmakers develop immobilization colon bacillus cell method and produce continuously L-Aspartic acid.1985 Nian， Nippon Mitsubishi Oil chemical companies utilize brevibacterium flavum (Brevibacterium flavum) free cell that fumaric acid is converted into L-Aspartic acid, and brevibacterium flavum is centrifugal rear reusable, has reduced production cost.
Early 1980s, China starts to develop L-Aspartic acid product, and the mid-80 adopts immobilized cell method to carry out production pilot scale, has carried out subsequently suitability for industrialized production.But because immobilized cell method processing unit input is many, technical requirements is high, makes the production cost of product relatively high.Along with being growing more intense of market competition, the Wang Xue of Nanjing University of Chemical Technology roots in 2000 etc. are developed free whole cell method, for suitability for industrialized production L-Aspartic acid.Free cell method is the main method of producing L-Aspartic acid at present.
Summary of the invention
Technical problem solved by the invention is to provide a kind of industry application that is suitable for, and bulk fermentation is produced the method for L-Aspartic acid.
L-Aspartic acid fermentation technique research in the past, all be confined in laboratory scale and small volume fermentor tank scale, although make some progress, once be applied to real industrialization, produce, large scale fermentation tank is produced, and corresponding fermentation condition all can not be obtained good effect.The present invention on this basis, studies a kind of to high yield L-Aspartic acid enzyme bacterial strain colon bacillus HY-05C, at 2m
3on the basis of tank maturing fermentation technique, amplify 10 times, by analog calculation and optimization of process conditions, determine 20m
3the optimal processing parameter of tank principal dimension parameter and fermentation, for 20m
3bulk fermentation is cultivated colon bacillus HY-05C, and fermented liquid transforms fumaric acid again and produces L-Aspartic acid.
For 20m
3bulk fermentation is cultivated colon bacillus and is transformed the method that fumaric acid is produced L-Aspartic acid again, is at tank diameter D=2200mm, the high H of tank shell portion
0in the large fermentation tank of=5280mm, fill liquid nutrient medium 90%(v/v), access colon bacillus seed liquor, inoculum size 0.12 ‰ (v/v), at ventilation 280m
3under the condition of/h, rotating speed 100rpm, tank pressure 0.05MPa, cultivate 13h, make number of viable 1.89 * 10
8the fermented liquid of cfu/ml, L-Aspartic acid enzyme activity 4725U/ml, then produce and obtain L-Aspartic acid with fermented liquid conversion fumaric acid.
Wherein said liquid nutrient medium (g/L): Dried Corn Steep Liquor Powder 13, sal epsom 0.2, potassium primary phosphate 1, sodium-chlor 1.45, fumaric acid 5; Glucose 5, adjusts pH to 6.5-7.0 with ammoniacal liquor.
The preparation method of wherein said colon bacillus seed liquor is: the shaking flask of 3L, and packing 700mL seed liquor substratum, after sterilizing, gets inclined-plane seed one ring and inoculates; 90rpm, cultivates 12 hours for 37 ℃ ± 1 ℃; Seed liquor substratum (g/L) wherein: fumaric acid 5; Glucose 5; Ammoniacal liquor 15ml: sodium chloride 5; Potassium primary phosphate 1; Sal epsom 0.2; Peptone 6.5; Dried Corn Steep Liquor Powder 6.5.
Wherein said fumaric acid is 16% fumaric acid solution.
L-Aspartic acid transformed bacteria HY-05C is intestinal bacteria, have another name called colon bacillus, that the L-Aspartic acid free cell conversion method of seed selection in Yantai Heng Yuan Biological Co., Ltd. early stage is produced bacterium (being documented in application number is CN201110382584.X, the applying date is: on November 25th, 2011, denomination of invention is: in the Chinese invention patent application of " colon bacillus and the application thereof of high yield L-Aspartic acid enzyme ", deposit number is CGMCC No.5450).
This bacterial strain has high L-Aspartic acid enzyme activity, transforms fumaric acid production L-Aspartic acid functional, for good basis has been established in the technical study of this project.On colon bacillus HY-05C nutrient agar plate, cultivate after 24 microspecies for 37 ℃, bacterium colony is faint yellow, and circle is translucent, and surface wettability is droplet-like, projection.Cell is thin rod-short, and size is (0.56 * 1.22～2.12) μ m, and Gram-negative, without gemma.
The colon bacillus HY-05C fermented liquid of applied culture of the present invention is produced for L-Aspartic acid, on average produces 1504 tons of ASPARTIC ACIDs per month, and yield is 106.05%.Quality product meets the requirement of national sector standard QB1118-91L-aspartic acid.
The present invention carries out 20m to high yield L-Aspartic acid enzyme bacterial strain colon bacillus HY-05C
3bulk fermentation research, has solved the gordian technique in bulk fermentation technique.The 20m3 bulk fermentation of key being: colon bacillus HY-05C, inoculum size is low, only needs directly inoculation 2.1L shake-flask seed liquid, and fermentation culture 13h, can obtain the fermented liquid with high L-Aspartic acid enzyme activity.Save the amplification culture step by step of seeding tank, avoided the microbiological contamination risk in seed amplification culture process, shortened fermentation period simultaneously, improved production efficiency.Fermentation culture 13h, fermented liquid viable count reaches 1.89 * 108cfu/ml, and L-Aspartic acid enzyme activity reaches 4725U/ml.The nutrient solution obtaining by bulk fermentation has been obtained unusual effect for L-Aspartic acid production, and production efficiency is significantly higher than similar level.The 20m of colon bacillus HY-05C
3bulk fermentation technology, can significantly improve production efficiency, in L-Aspartic acid conversion method is produced, has broad prospect of application.
Accompanying drawing explanation
The impact of Fig. 1 ventilation on fermentation;
The impact of Fig. 2 different vaccination amount on fermentation;
The impact that Fig. 3 inoculum size is lived on enzyme;
Figure 42 0m
3tank fermented bacterium growth curve;
Figure 52 0m
36 batches of zymophyte bulk-growth curves of tank;
Figure 62 0m
36 batches of fermentation broth enzymes of tank are lived.
Embodiment
For further illustrating the present invention, illustrate with the following Examples:
1, bacterial classification:
Colon bacillus (Escherichia coli), be numbered: HY-05C, enterprise is own, and (being documented in application number is CN201110382584.X, the applying date is: on November 25th, 2011, denomination of invention is: in the Chinese invention patent application of " colon bacillus and the application thereof of high yield L-Aspartic acid enzyme ", deposit number is CGMCC No.5450).
2, solution and substratum:
Solution
(1) 16% fumaric acid solution: fumaric acid 40g, be dissolved in 200ml pure water, with ammoniacal liquor, adjust pH7.5, with pure water, be settled to 250ml.
(2) 2% Adlerikas: take 2g sal epsom (analytical pure), be settled to 100ml after dissolving with distilled water.
(3) 1:10 dilute sulphuric acid: get 1 part of 98% vitriol oil, be dissolved in 10 parts of distilled water.
Substratum
(1) preservation slant medium (g/L): sodium chloride 5; Potassium primary phosphate 5; 2% (W/V) Adlerika 10ml; Peptone 7; Extractum carnis 10; Agar 22.
(2) produce slant medium (g/L): fumaric acid 10; Ammoniacal liquor 10ml: sodium chloride 5; Potassium primary phosphate 1; 2% (W/V) Adlerika 10ml; Peptone 7; Extractum carnis 10; Agar 20.
(3) seed liquor substratum (g/L): fumaric acid 5; Glucose 5; Ammoniacal liquor 15ml: sodium chloride 5; Potassium primary phosphate 1; Sal epsom 0.2; Peptone 6.5; Dried Corn Steep Liquor Powder 6.5.
(4) fermention medium (g/L): Dried Corn Steep Liquor Powder 13, sal epsom 0.2, potassium primary phosphate 1, sodium-chlor 1.45, fumaric acid 5; Glucose 5, adjusts pH to 6.5-7.0 with ammoniacal liquor.
3, seed culture:
The shaking flask of 3L, packing 700mL seed liquor substratum, after sterilizing, gets production inclined-plane seed one ring and inoculates.90rpm, cultivates 12 hours for 37 ℃ ± 1 ℃.
4, L-Aspartic acid enzyme activity determination:
Enzyme is lived and is defined: at 45 ℃, under pH value 7.5 conditions, the enzyme amount of conversion 1 micromole's fumaric acid substrate per hour is an enzyme activity unit U.
16% fumaric acid solution 250ml is packed in triangular flask, add fermented liquid 20ml, then add 2ml2% Adlerika, shake up.Triangular flask is put into the water-bath of 45 ℃, transformed, and sample respectively detection at 0h, 1h.
Get the H of 1:10
2sO
4solution 35ml puts into 250ml triangular flask, then sucks 0.2ml conversion fluid and add wherein, is heated to 75-80 ℃, uses 0.02M KMnO
4solution is titrated to micro-red, colour-fast in half a minute, is terminal.Read the KMnO of consumption
4volume milliliter number, 0h sample consumes volume and is designated as V
empty, reaction 1h terminal consumes volume and is designated as V
sample, calculate as follows bio-transformation enzyme activity:
Fumaric acid and KMnO
4chemical reaction:
C
4H
4O
4+2KMnO
4+3H
2SO
4→HCOOH+3CO
2+K
2SO
4+2MnSO
4+4H
2O
1ml0.02mol/L KMnO4～1.16mg fumaric acid
5C
4h
4o
4+ 4KMnO
4+ 6H
2sO
4→ 5C
4h
4o
6(tartrate)+2K
2sO
4+ 4MnSO
4+ 6H
2o
1ml0.02mol/L KmnO4～2.9mg fumaric acid
Get (1.16+2.9)/2 ≈ 2mg fumaric acid
Note: the molecular weight of 116-fumaric acid;
2-every consumption 1ml0.02M potassium permanganate is equivalent to 2mg fumaric acid;
272-reaction cumulative volume 272ml;
20-add 20ml fermented liquid;
1 hour 1-reaction times;
0.2-get 0.2ml reaction solution to measure.
5,20m
3tank fermentation technology is optimized and conclusion:
(1) air flow affects enzymatic production
Air flow major effect fermented liquid dissolved oxygen level, dissolved oxygen height has material impact to growth and enzyme activity.The amplification of fermentor tank at present is still carried out in experience or semiempirical situation.This research is with 2m
3the service data of fermentor tank is basis, take geometricsimilarity as prerequisite, and to take the volume transmission coefficient of oxygen be index, has determined 20m
3the mixing speed of tank and air flow, mixing speed is 100rpm, air flow is 180m
3/ h.When carrying out amplification culture test, ventilation is 180m
3/ h, thalli growth is slow.Therefore, fixedly mixing speed 100rpm, is optimized air flow, has studied respectively air flow and be 180,230,280,300,320m
3during/h, the impact on thalli growth and product enzyme.
When rotating speed 100rpm, along with the increase (180-280m of air flow
3/ h), the dissolved oxygen of fermention medium improves, and thalli growth takes a turn for the better thereupon, and enzyme activity also increases thereupon, but work as air flow, is greater than 280m
3after/h, the growth of thalline maintains comparatively stable level, and the raising that enzyme is lived is also not obvious, finally determines that best air flow is 280m
3/ h.
Table 1 ventilation is on fermentation and enzyme activity impact
| Air flow (m 3/h） | 180 | 230 | 280 | 300 | 320 |
| Fermentation time (h) | 20 | 18 | 14 | 14 | 14 |
| OD value | 0.851 | 0.959 | 1.031 | 1.012 | 1.021 |
| Enzyme (U/ml) alive | 2061 | 3557 | 4769 | 4737 | 4680 |
(2) inoculum size affects enzymatic production
The size of inoculum size is decided by produce the speed of bacterial classification growth and breeding in fermentor tank, adopts larger inoculum size can shorten the time that in fermentor tank, thalline breeding peaks, and the formation of product is arrived in advance, and can reduce the growth machine meeting of miscellaneous bacteria.But inoculum size is excessive or too small, all can affect fermentation.Cross conference and cause that dissolved oxygen is not enough, the expression that affects enzyme is synthetic, and inoculum size conference is also uneconomical; The too small meeting Extending culture time of inoculum size, the productivity of reduction fermentor tank.At 20m
3the thalline production of inoculum size and the impact of producing enzyme in tank, have been investigated.
Result in Fig. 2 shows, the growth of inoculum size major effect, and then affect the expression of enzyme.Inoculum size 0.02 ‰ and 0.06 ‰ is too low, be unfavorable for the expression that thalli growth and enzyme are lived, along with inoculum size increases, thalli growth and enzyme are lived and are not significantly increased thereupon, but maintaining a metastable level, test is final determines that best inoculum size is 0.12 ‰.
(3) impact of defoamer on enzymatic production
The large tank of microorganism ventilates and stirs in fermentation, and foam is the important factor that impact is produced, and the large dissolved oxygen of foam volume causes fermented liquid excessive, and pollution microbes has a big risk.Therefore in fermentative production, the normal defoamer that adds suppresses foam generation.At 2m
3during tank, every tank defoamer dosage is at 150ml, 20m
3during tank, defoamer dosage has been carried out to suitable minimizing.Result in table 2 shows, when defoamer dosage is more than or equal to the every tank of 1350ml, can effectively eliminate issuable foam in fermenting process.When the every tank of 1200ml, there is slight foaming.
Table 2 fermentation defoaming agent dosage
| Defoamer dosage (ml/ tank) | 1200 | 1350 | 1400 | 1450 | 1500 |
| OD value | 1.029 | 1.02 | 1.019 | 0.976 | 0.957 |
| Enzyme (U/ml) alive | 4645 | 4759 | 4742 | 4717 | 4811 |
| Foam degree | + | - | - | - | - |
+/-:+there is certain foaming ,-expression is not bubbled;
(4) growth curve under optimum process condition
After having determined best technological condition for fermentation, determined, measured 20m
3tank fermented bacterium growth curve.Growth and product enzyme curve are as Fig. 4, and after result shows cultivation 11h, pH reaches more than 8.0, and OD value and enzyme activity are substantially constant after 13h, and 13h enzyme activity reaches 4686U/ml.So 20m
3tank fermented incubation time is defined as 13h, with 2m
3tank fermentation is consistent.In actual production, can change judgement fermentation termination according to OD value and pH.
(5) multiple batches of fermentation proof test
20m
3the fermentation of 6 batches has been carried out in the fermentation stability test of tank altogether, has measured the OD value of 13h fermented liquid, pH, viable count and enzyme activity.
Continuous 6 batches of 20m
3fermentor tank scale-up, liquid amount is 18000L, inoculum size is 2.1L(0.12 ‰), culture temperature is 37 ℃, and tank pressure is 0.05Mpa, and ventilation is 280m
3/ h, rotating speed 100rpm, the parameter detailed datas such as the fermentation time of fermentor tank, number of viable, pH value, enzyme work are in Table 3.
From table 3 and Fig. 5,6 can find out, the fermentation period of 6 tanks that continuously ferment is 13h.The OD value of thalline all reaches 1.0, and growth shows a rising trend between whole yeast phase.In the time of thalli growth, follow L-Aspartic acid enzymic synthesis, pH value rising, at fermentation termination all in 8.0 left and right.Fermented liquid L-Aspartic acid enzyme activity mean value is 4725U/ml, viable count 1.89 * 10
8cfu/ml, a little more than original 2m
3ferment tank level.20m
36 batches of fermentation tests of tank are stable, and it is respond well that fermented liquid is used for transforming fumaric acid production L-Aspartic acid.
Table 320m
3the tank 6 batches of parameters of continuously fermenting
| |
1 | 2 | 3 | 4 | 5 | 6 |
| |
8 | 8 | 8 | 8 | 8 | 8 |
| Fermentation time (h) | 13 | 13 | 13 | 13 | 13 | 13 |
| Number of viable (cfu/ml) | 1.91×10 8 | 1.92×10 8 | 1.89×10 8 | 1.87×10 8 | 1.90×10 8 | 1.87×10 8 |
| Enzyme is lived | 4737 | 4797 | 4752 | 4708 | 4667 | 4691 |
Bulk fermentation technology is applied to L-Aspartic acid and produces, and continuous and stable production is 7 months, and L-Aspartic acid production data sees the following form.
Continuous 7 months L-Aspartic acid production datas (2012.10-2013.4) of table 4
| Month | Fumaric acid feed intake (ton) | Produce L-Aspartic acid (ton) | Recovery rate (%) |
| In October, 2012 | 1363.470 | 1357.365 | 107.43 |
| In November, 2012 | 1307.585 | 1407.253 | 107.62 |
| In December, 2012 | 1369.932 | 1460.210 | 106.59 |
| In January, 2013 | 1408.475 | 1480.410 | 105.11 |
| In February, 2013 | 1373.365 | 1443.408 | 105.10 |
| In March, 2013 | 1577.710 | 1656.282 | 104.98 |
| In April, 2013 | 1636.210 | 1721.293 | 105.53 |
| Amount to | 10036.75 | 10526.22 | / |
Note: recovery rate is L-asparagus fern ammonia/fumaric acid
Year April in October, 2012 to 2013, the 20m3 bulk fermentation technology of colon bacillus HY-05C is applied to L-Aspartic acid and produces, 10036.75 tons of fumaric acid feed intake for 7 months altogether, obtain 10526.22 tons of L-Aspartic acids, minimum yield is 104.98%, the highest yield is 107.62%, and average yield is 106.05%.Bulk fermentation technology is applied to L-Aspartic acid and produces, and has realized the industrial scale of producing 1504 tons per month.L-Aspartic acid product meets the requirement of national sector standard QB1118-91L-aspartic acid.
The present invention is directed to high yield L-Aspartic acid enzyme bacterial strain colon bacillus HY-05C, on the basis of 2m3 tank maturing fermentation technique, amplify 10 times, by analog calculation and optimization of process conditions, determine the optimal processing parameter of 20m3 tank principal dimension parameter and fermentation, for 20m3 bulk fermentation, cultivated colon bacillus HY-05C.20m3 bulk fermentation is cultivated colon bacillus HY-05C, and fermented liquid viable count reaches 1.89 * 108cfu/ml, and L-Aspartic acid enzyme activity reaches 4725U/ml.Fermented liquid is used for transforming fumaric acid production L-Aspartic acid and obtains good result.20m3 fermentor tank scale, colon bacillus HY-05C saves seeding tank amplification culture step by step, and low inoculum size level, directly inoculates shake-flask seed liquid, and fermentation culture 13h can obtain the fermented liquid with high L-Aspartic acid enzyme activity, and enzyme activity reaches 4725U/ml.Save the amplification culture step by step of seeding tank, can avoid the microbiological contamination risk in seed amplification culture process, can shorten fermentation period, enhance productivity simultaneously.This technology is applied to L-Aspartic acid and produces, and continuous and steady operation is 7 months, and accumulative total is produced 10526.22 tons of ASPARTIC ACIDs, on average produces 1504 tons of ASPARTIC ACIDs per month, and yield is 106.05%.Product meets the requirement of national sector standard QB1118-91L-aspartic acid.
Above-described embodiment is described the preferred embodiment of the present invention; not scope of the present invention is limited; design under the prerequisite of spirit not departing from the present invention; various distortion and improvement that the common engineering technical personnel in this area make technical scheme of the present invention, all should fall in the definite protection domain of claims of the present invention.
Claims (4)
1. for 20m
3bulk fermentation is cultivated colon bacillus and is transformed the method that fumaric acid is produced L-Aspartic acid again, it is characterized in that: at tank diameter D=2200mm, the high H of tank shell portion
0in the large fermentation tank of=5280mm, fill liquid nutrient medium 90%(v/v), access colon bacillus seed liquor, inoculum size 0.12 ‰ (v/v), at ventilation 280m
3under the condition of/h, rotating speed 100rpm, tank pressure 0.05MPa, cultivate 13h, make number of viable 1.89 * 10
8the fermented liquid of cfu/ml, L-Aspartic acid enzyme activity 4725U/ml, then produce and obtain L-Aspartic acid with fermented liquid conversion fumaric acid.
2. method according to claim 1, is characterized in that: described liquid nutrient medium (g/L): Dried Corn Steep Liquor Powder 13, sal epsom 0.2, potassium primary phosphate 1, sodium-chlor 1.45, fumaric acid 5; Glucose 5, adjusts pH to 6.5-7.0 with ammoniacal liquor.
3. method according to claim 1, is characterized in that: the preparation method of described colon bacillus seed liquor is: the shaking flask of 3L, and packing 700mL seed liquor substratum, after sterilizing, gets inclined-plane seed one ring and inoculates; 90rpm, cultivates 12 hours for 37 ℃ ± 1 ℃; Seed liquor substratum (g/L) wherein: fumaric acid 5; Glucose 5; Ammoniacal liquor 15ml: sodium chloride 5; Potassium primary phosphate 1; Sal epsom 0.2; Peptone 6.5; Dried Corn Steep Liquor Powder 6.5.
4. method according to claim 1, is characterized in that: the fumaric acid solution that described fumaric acid is 16%.
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Cited By (4)
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| CN104593306A (en) * | 2015-02-04 | 2015-05-06 | 烟台恒源生物股份有限公司 | High-density culture method of escherichia coli strains HY-05C |
| CN106588682A (en) * | 2016-10-28 | 2017-04-26 | 张家港市华昌药业有限公司 | New preparation method of ethyl aspartate hydrochloride |
| CN109355327A (en) * | 2018-12-18 | 2019-02-19 | 山东金洋药业有限公司 | A kind of L- hydroxyproline bacteria suspension is into tank inoculation method |
| CN111893077A (en) * | 2020-08-24 | 2020-11-06 | 河南金百合生物科技股份有限公司 | Large-scale production method of clostridium butyricum |
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|---|---|---|---|---|
| CN104593306A (en) * | 2015-02-04 | 2015-05-06 | 烟台恒源生物股份有限公司 | High-density culture method of escherichia coli strains HY-05C |
| CN104593306B (en) * | 2015-02-04 | 2017-06-23 | 烟台恒源生物股份有限公司 | A kind of E bacterial strain HY 05C high-density cultivation methods |
| CN106588682A (en) * | 2016-10-28 | 2017-04-26 | 张家港市华昌药业有限公司 | New preparation method of ethyl aspartate hydrochloride |
| CN109355327A (en) * | 2018-12-18 | 2019-02-19 | 山东金洋药业有限公司 | A kind of L- hydroxyproline bacteria suspension is into tank inoculation method |
| CN111893077A (en) * | 2020-08-24 | 2020-11-06 | 河南金百合生物科技股份有限公司 | Large-scale production method of clostridium butyricum |
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Denomination of invention: Method for production of L-aspartic acid by fermentation of Escherichia coli in 20 m3large tank Effective date of registration: 20221228 Granted publication date: 20150722 Pledgee: Yantai Longkou sub branch of Rizhao Bank Co.,Ltd. Pledgor: YANTAI HENGYUAN BIOENGINEERING Co.,Ltd. Registration number: Y2022980028953 |