CN109182423A - Promote the method for Escherichia coli fermentation production poly sialic acid - Google Patents
Promote the method for Escherichia coli fermentation production poly sialic acid Download PDFInfo
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Abstract
A method of promote Escherichia coli fermentation to produce poly sialic acid, is related to field of fermentation engineering.The promotion Escherichia coli fermentation produces the method for poly sialic acid the following steps are included: Escherichia coli are seeded to fermentation medium cultivation and fermentation, isolated poly sialic acid；When cultivation and fermentation, controlling the temperature between cultivation and fermentation 0-48h is 40-42 DEG C, and the temperature after control cultivation and fermentation 48h is 36-38 DEG C.The method of promotion Escherichia coli fermentation production poly sialic acid can effectively improve fermentation level, combined coefficient, the carbon source transformation efficiency of poly sialic acid.
Description
Technical field
The present invention relates to a kind of field of fermentation engineering, and in particular to a kind of promotion Escherichia coli fermentation produces poly sialic acid
Method.
Background technique
N-acetyl-neuraminate (N-acetylneuraminic acid) Neu5Ac is the first contact of cellular informatics transmitting
Site, molecular structure has diversity, therefore Neu5Ac participation cell recognition, signal transduction, tumour generation, fertilization etc. are multiple
Physiology course, Neu5Ac are also adjustable the anti-inflammatory activity of IgG, enhance infant immunity, influence integrality, the infiltration of nerve cell
Property and activity, promote the development of infant brain, so the production of N-acetyl-neuraminate causes more concern and research.
At present the production method of N-acetyl-neuraminate include: natural product extraction, chemical synthesis, biological enzyme urge conversion,
Genetic engineering bacterium direct fermentation and poly sialic acid fermentation hydrolysis method.Since the content of Neu5Ac in most natural products is low,
And component is extremely complex, therefrom extracts Neu5Ac along with complicated process, the rate of recovery is low, is difficult to meet wanting for large-scale production
It asks.And chemical synthesis is due to the operation such as cumbersome radical protection, deprotection and the presence of chiral photo-isomerisation by-product, the party
Method can not also be applied to industrial production.Biological enzyme is urged conversion and genetic engineering bacterium to be related to transgenosis problem and is also difficult to through food phase
Law approval is closed, it is even more impossible to be applied to infantile health field.Only poly sialic acid fermentation hydrolysis method is since it belongs to natural production
Object, consumer are easier to approve, and are approved by the Chinese authority, industrialization value with higher.
Poly sialic acid (Polysialic acid, PSA) is N-acetyl-neuraminate with the key connection of α -2,8 and (or) α -2,9
Homopolymer, be the main component of the capsular polysaccharide of a small number of bacteriums.Nineteen fifty-seven, Barry and Goebel are first in E.coli K235
Middle discovery poly sialic acid, later people have found poly sialic acid in other bacterial strains in succession.But the fermentation water of current poly sialic acid
Flat, combined coefficient and carbon source transformation efficiency are lower, and the yield of poly sialic acid PSA is only 5~6g/L when fermenting and producing 30-40h, lead
It causes fermentation costs higher, and then limits the application of N-acetyl-neuraminate.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of promotion Escherichia coli fermentation production poly sialic acid, can be effective
Raising poly sialic acid fermentation level, combined coefficient and carbon source transformation efficiency.
The present invention solves its technical problem and adopts the following technical solutions to realize.
A method of promote Escherichia coli fermentation to produce poly sialic acid comprising following steps: Escherichia coli are inoculated with
To fermentation medium cultivation and fermentation, isolated poly sialic acid；When cultivation and fermentation, the temperature between cultivation and fermentation 0-48h is controlled
Degree is 40-42 DEG C, and the temperature after control cultivation and fermentation 48h is 36-38 DEG C.
Further, in a preferred embodiment of the present invention, when above-mentioned cultivation and fermentation, controlling initial ventilatory capacity is 0.8-
1.2vvm, initial speed 150-250rpm；Dissolved oxygen content is adjusted after inoculation in 3mg/L or more.
Further, in a preferred embodiment of the present invention, when above-mentioned cultivation and fermentation, fermented and cultured is seeded to from Escherichia coli
The 8h that base rises, cetyl trimethylammonium bromide is added into fermentation medium, reaches cetyl trimethylammonium bromide
150-250mg/L。
Further, in a preferred embodiment of the present invention, when above-mentioned cultivation and fermentation, when carbon source has consumed in fermentation medium
Afterwards, with the rate supplement sorbierite of 1-4g/ (Lh) into fermentation medium.
It further, in a preferred embodiment of the present invention, the use of ammonia spirit control pH is 6.0- when above-mentioned cultivation and fermentation
6.5。
Further, in a preferred embodiment of the present invention, above-mentioned fermentation medium includes following component: the mountain of 15-25g/L
Pears alcohol, the Dried Corn Steep Liquor Powder of 2-4g/L, the dipotassium hydrogen phosphate of 3-7g/L, the magnesium sulfate of 0.3-0.8g/L, 4-8g/L ammonium chloride,
N-acetylglucosamine, the B family vitamin solution of 0.5-2mL/L and the trace element solution of 0.5-2mL/L of 4-8g/L.
Further, in a preferred embodiment of the present invention, above-mentioned B family vitamin solution includes following component: 0.3-0.6g/
The VB of L1, 3-3.5g/L VB5, 0.001-0.01g/L VBH and 0.1-0.2g/L VB12。
Further, in a preferred embodiment of the present invention, above-mentioned trace element solution includes following component: 1-4g/L's
FeSO4, 0.01-0.1g/L KIO3, 0.5-1g/L MnCl2, 0.1-0.5g/L CoCl2, 0.01-0.1g/L CrCl3、
The ZnSO of 0.01-0.2g/L4, 0.01-0.05g/L Na2MoO4And the H of 1-2g/L3BO3。
Further, in a preferred embodiment of the present invention, above-mentioned fermentation medium further includes the sucrose fat of 0.1-1mL/L
Acid esters.
Further, in a preferred embodiment of the present invention, the deposit number of above-mentioned Escherichia coli is CCTCC NO:M
2018103。
The beneficial effect of the Escherichia coli of the embodiment of the present invention and its application in fermenting and producing poly sialic acid is: this hair
The method for the promotion Escherichia coli fermentation production poly sialic acid that bright embodiment provides is: Escherichia coli are seeded to fermentation medium
Cultivation and fermentation, isolated poly sialic acid；When cultivation and fermentation, controlling the temperature between cultivation and fermentation 0-48h is 40-42 DEG C,
Temperature after control cultivation and fermentation 48h is 36-38 DEG C.The method of promotion Escherichia coli fermentation production poly sialic acid can have
Fermentation level, combined coefficient and the carbon source transformation efficiency of the raising poly sialic acid of effect.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is that Escherichia coli fermentation produces poly sialic acid poly sialic acid under the different fermentations time in the embodiment of the present invention 1
The curve graph of content and absorbance；
Fig. 2 is that Escherichia coli fermentation produces poly sialic acid poly sialic acid under the different fermentations time in comparative example 1 of the present invention
The curve graph of content and absorbance；
Fig. 3 is that Escherichia coli fermentation produces poly sialic acid poly sialic acid under the different fermentations time in comparative example 2 of the present invention
The curve graph of content and absorbance.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
The application to the Escherichia coli of the embodiment of the present invention and its in fermenting and producing poly sialic acid carries out specifically below
It is bright.
The present invention provides a kind of methods of promotion Escherichia coli fermentation production poly sialic acid comprising following steps:
Escherichia coli are seeded to fermentation medium cultivation and fermentation, isolated poly sialic acid；When cultivation and fermentation, control training
Temperature between hair care ferment 0-48h is 40-42 DEG C, and the temperature after control cultivation and fermentation 48h is 36-38 DEG C.Preferably, will
Escherichia coli are cultivated to obtain seed liquor, and then seed liquor is seeded in fermentation medium and is cultivated.
Application of the Escherichia coli provided in an embodiment of the present invention in fermenting and producing poly sialic acid is by by the large intestine bar
Bacterium is seeded to fermentation medium cultivation and fermentation and obtains poly sialic acid, by using stage alternating temperature control fermentation culture method,
Cultivation and fermentation temperature is controlled between the 0-48h of cultivation and fermentation at 40-42 DEG C, and culture is controlled after the 48h of cultivation and fermentation
Fermentation temperature is 36-38 DEG C, can effectively improve carbon source forward rate, PSA fermentation level and combined coefficient.To improve poly- saliva
The production efficiency of liquid acid.
In a kind of preferred scheme of the present invention, when cultivation and fermentation, controlling initial ventilatory capacity is 0.8-1.2vvm, initial to turn
Speed is 150-250rpm；Dissolved oxygen content is adjusted after inoculation in 3mg/L or more.By controlling fermentation process after inoculation Escherichia coli
In ventilation index, can guarantee Escherichia coli breeding control environment, promote Escherichia coli fermentation produce poly sialic acid.
In a kind of preferred scheme of the present invention, when cultivation and fermentation, from Escherichia coli are seeded to fermentation medium the
8h adds cetyl trimethylammonium bromide into fermentation medium, and cetyl trimethylammonium bromide is made to reach 150-
250mg/L.Cetyl trimethylammonium bromide is added during cultivation and fermentation, cell pod membrane can be promoted to fall off, to improve
The productivity of poly sialic acid.
In a kind of preferred scheme of the present invention, when cultivation and fermentation, after carbon source has consumed in fermentation medium, with 1-4g/
(Lh) rate supplement sorbierite is into fermentation medium.By continuing to add carbon source into fermentation medium, can guarantee
Constantly fermentation generates poly sialic acid after Escherichia coli breeding.
It the use of ammonia spirit control pH is 6.0-6.5 when cultivation and fermentation in a kind of preferred scheme of the present invention.It is preferred that
PH is controlled using the ammonia spirit that mass percent is 5-10%.PH value by controlling cultivation and fermentation can promote Escherichia coli
Growth and breeding, thus effectively improve poly sialic acid productivity.
In a kind of preferred scheme of the present invention, fermentation medium includes following component: sorbierite, the 2- of 15-25g/L
The Dried Corn Steep Liquor Powder of 4g/L, the dipotassium hydrogen phosphate of 3-7g/L, the magnesium sulfate of 0.3-0.8g/L, the ammonium chloride of 4-8g/L, 4-8g/L
N-acetylglucosamine, the B family vitamin solution of 0.5-2mL/L and the trace element solution of 0.5-2mL/L.Wherein,
Preferably, B family vitamin solution includes following component: the VB of 0.3-0.6g/L1, 3-3.5g/L VB5, 0.001-0.01g/L
The VB of VBH and 0.1-0.2g/L12.It is furthermore preferred that trace element solution includes following component: the FeSO of 1-4g/L4、0.01-
The KIO of 0.1g/L3, 0.5-1g/L MnCl2, 0.1-0.5g/L CoCl2, 0.01-0.1g/L CrCl3、0.01-0.2g/L
ZnSO4, 0.01-0.05g/L Na2MoO4And the H of 1-2g/L3BO3.By configuring the fermentation medium of suitable component, energy
The breeding for enough promoting Escherichia coli quick, so that promoting Escherichia coli to carry out fermentation to fermentation medium generates poly sialic acid.
It further include the sucrose fatty ester of 0.1-1mL/L in fermentation medium in a kind of preferred scheme of the present invention.It is logical
It crosses and sucrose fatty ester is added into fermentation medium as defoaming agent, be able to suppress foam generation, avoid producing in fermentation process
Raw a large amount of foam influences the progress of fermentation process, guarantees the progress of fermentation stability to produce to obtain poly sialic acid.
In a kind of preferred scheme of the present invention, Escherichia coli are CCTCC NO:M's 2018103 selected from deposit number
Escherichia coli.The Escherichia coli have been filed on March 6th, 2018 to the China of Wuhan City, Hubei Province Wuchang District Bayi Road Luo Jia Shan
Type Tissue Collection (CCTCC) preservation, deposit number CCTCC NO:M 2018103；Taxology name:
Escherichia coli CASOV-8。
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The embodiment of the invention provides it is a kind of using Escherichia coli fermentation production poly sialic acid method, mainly include with
Lower step:
S11, take the strain streak inoculation of the Escherichia coli (deposit number CCTCC NO:M 2018103) frozen to solid
Culture obtains single colonie at 37 DEG C on LB culture medium；Used Escherichia coli have been filed on March 6th, 2018 to Hubei Province
China typical culture collection center (CCTCC) preservation of wuchang, wuhan area Bayi Road Luo Jia Shan；Wherein, solid LB media
Contain following components: peptone 10g/L, yeast extract powder 5g/L, sodium chloride 10g/L, agar 20g/L；Solid LB media
PH is 7.0.
S12, picking single colonie are inoculated into the triangular flask of the LB seed culture medium of liquid containing 50mL, in the stirring of 200rpm
Overnight incubation at rate and 37 DEG C, obtains primary seed solution；Wherein, liquid LB seed culture medium contains following components: peptone
10g/L, yeast extract powder 5g/L, sodium chloride 10g/L；The pH of liquid LB seed culture medium is 7.0.
S13, primary seed solution is connected to the three of the LB seed culture medium of liquid containing 50mL by the inoculum concentration of 1% percent by volume
In the bottle of angle, 6h is cultivated at the stirring rate of 200rpm and 37 DEG C, obtains secondary seed solution.
S14, the fermentation medium that 3L is packed into 5L fermentor, 100mL secondary seed solution is seeded in fermentor and is trained
It supports；Wherein, fermentation medium contains following components: sorbierite 20g/L, Dried Corn Steep Liquor Powder 3g/L, dipotassium hydrogen phosphate 5g/L, sulfuric acid
Magnesium 0.5g/L, ammonium chloride 6g/L, N-acetylglucosamine 6g/L, B family vitamin solution 1mL/L and trace element solution
1mL/L.Wherein, B family vitamin solution contains following components: the VB of 0.5g/L1, 3.2g/L VB5, 0.006g/L VBH and
The VB of 0.15g/L12；Trace element solution contains following components: the FeSO of 2g/L4, 0.06g/L KIO3, the MnCl of 0.8g/L2、
The CoCl of 0.2g/L2, 0.08g/L CrCl3, 0.09g/L ZnSO4, 0.03g/L Na2MoO4And the H of 1.75g/L3BO3。
In S15, incubation, controlling the temperature between cultivation and fermentation 0-48h is 40 DEG C, the temperature after cultivation and fermentation 48h
Degree is 37 DEG C, and the initial ventilatory capacity after control inoculation is 1vvm, initial speed 200rpm；Dissolved oxygen content DO is adjusted after inoculation
It the use of pH in the ammonium hydroxide position fermentor of mass concentration 10% is 6.4 in fermentation process for 3mg/L, the to ferment after inoculation
Six alkyl trimethyl ammonium bromides are added into fermentation medium by 8h, reach cetyl trimethylammonium bromide in fermentation medium
200mg/L, after carbon source has consumed in fermentation medium, with the rate supplement sorbierite of 2.5g/ (Lh) into fermentation medium.
Tank is put after S16, fermented and cultured 72h takes tunning, isolated poly sialic acid.
Embodiment 2
The embodiment of the invention provides it is a kind of using Escherichia coli fermentation production poly sialic acid method, mainly include with
Lower step:
S21, take the strain streak inoculation of the Escherichia coli (deposit number CCTCC NO:M 2018103) frozen to solid
Culture obtains single colonie at 37 DEG C on LB culture medium；Wherein, solid LB media contains following components: peptone 10g/L, ferment
Female extract powder 5g/L, sodium chloride 10g/L, agar 20g/L；The pH of solid LB media is 7.0.
S22, picking single colonie are inoculated into the triangular flask of the LB seed culture medium of liquid containing 50mL, in the stirring of 200rpm
Overnight incubation at rate and 37 DEG C, obtains primary seed solution；Wherein, liquid LB seed culture medium contains following components: peptone
10g/L, yeast extract powder 5g/L, sodium chloride 10g/L；The pH of liquid LB seed culture medium is 7.0.
S23, primary seed solution is connected to the three of the LB seed culture medium of liquid containing 50mL by the inoculum concentration of 1% percent by volume
In the bottle of angle, 6h is cultivated at the stirring rate of 200rpm and 37 DEG C, obtains secondary seed solution.
S24, the fermentation medium that 3L is packed into 5L fermentor, 100mL secondary seed solution is seeded in fermentor and is trained
It supports；Wherein, fermentation medium contains following components: sorbierite 25g/L, Dried Corn Steep Liquor Powder 4g/L, dipotassium hydrogen phosphate 6g/L, sulfuric acid
Magnesium 0.6g/L, ammonium chloride 8g/L, N-acetylglucosamine 8g/L, sucrose fatty ester 0.5ml/L, B family vitamin solution
1.5mL/L and trace element solution 1.5mL/L.Wherein, B family vitamin solution contains following components: the VB of 0.6g/L1、
The VB of 3.5g/L5, 0.008g/L VBH and 0.18g/L VB12；Trace element solution contains following components: 3g/L's
FeSO4, 0.08g/L KIO3, the MnCl of 0.9g/L2, 0.3g/L CoCl2, 0.09g/L CrCl3, 0.1g/L ZnSO4、
The Na of 0.05g/L2MoO4And the H of 2g/L3BO3。
In S25, incubation, controlling the temperature between cultivation and fermentation 0-48h is 40 DEG C, the temperature after cultivation and fermentation 48h
Degree is 37 DEG C, and the initial ventilatory capacity after control inoculation is 1.2vvm, initial speed 200rpm；Dissolved oxygen content is adjusted after inoculation
DO is 3.5mg/L, and it is 6.4 that pH in the ammonium hydroxide position fermentor of mass concentration 10% is used in fermentation process, works as fermentation medium
After middle carbon source has consumed, with the rate supplement sorbierite of 3g/ (Lh) into fermentation medium.
Tank is put after S26, fermented and cultured 72h takes tunning, isolated poly sialic acid.
Embodiment 3
The embodiment of the invention provides it is a kind of using Escherichia coli fermentation production poly sialic acid method, mainly include with
Lower step:
S31, take the strain streak inoculation of the Escherichia coli (deposit number CCTCC NO:M 2018103) frozen to solid
Culture obtains single colonie at 37 DEG C on LB culture medium；Wherein, solid LB media contains following components: peptone 10g/L, ferment
Female extract powder 5g/L, sodium chloride 10g/L, agar 20g/L；The pH of solid LB media is 7.0.
S32, picking single colonie are inoculated into the triangular flask of the LB seed culture medium of liquid containing 50mL, in the stirring of 200rpm
Overnight incubation at rate and 37 DEG C, obtains primary seed solution；Wherein, liquid LB seed culture medium contains following components: peptone
10g/L, yeast extract powder 5g/L, sodium chloride 10g/L；The pH of liquid LB seed culture medium is 7.0.
S33, primary seed solution is connected to the three of the LB seed culture medium of liquid containing 50mL by the inoculum concentration of 1% percent by volume
In the bottle of angle, 6h is cultivated at the stirring rate of 200rpm and 37 DEG C, obtains secondary seed solution.
S34, the fermentation medium that 3L is packed into 5L fermentor, 100mL secondary seed solution is seeded in fermentor and is trained
It supports；Wherein, fermentation medium contains following components: sorbierite 18g/L, Dried Corn Steep Liquor Powder 2.5g/L, dipotassium hydrogen phosphate 4g/L, sulphur
Sour magnesium 0.4g/L, ammonium chloride 5g/L, N-acetylglucosamine 5g/L, B family vitamin solution 1mL/L and trace element solution
1mL/L.Wherein, B family vitamin solution contains following components: the VB of 0.4g/L1, 3.0g/L VB5, 0.005g/L VBH and
The VB of 0.12g/L12；Trace element solution contains following components: the FeSO of 1.5g/L4, 0.05g/L KIO3, 0.6g/L's
MnCl2, 0.15g/L CoCl2, 0.06g/L CrCl3, 0.075g/L ZnSO4, 0.02g/L Na2MoO4And 1.6g/L
H3BO3。
In S35, incubation, controlling the temperature between cultivation and fermentation 0-48h is 40 DEG C, the temperature after cultivation and fermentation 48h
Degree is 37 DEG C, and the initial ventilatory capacity after control inoculation is 0.8vvm, initial speed 200rpm；Dissolved oxygen content is adjusted after inoculation
DO is 4mg/L, and it is 6.4 that pH in the ammonium hydroxide position fermentor of mass concentration 10% is used in fermentation process, when in fermentation medium
After carbon source has consumed, with the rate supplement sorbierite of 2g/ (Lh) into fermentation medium.
Tank is put after S36, fermented and cultured 72h takes tunning, isolated poly sialic acid.
Comparative example 1
Large intestine is used in the method and embodiment 1 using Escherichia coli fermentation production poly sialic acid that this comparative example provides
The method that bacillus fermentation produces poly sialic acid is roughly the same, and difference is, the incubation in the step S15 of this comparative example, control
Temperature between cultivation and fermentation 0-48h processed is 37 DEG C, and the temperature after cultivation and fermentation 48h is 37 DEG C.
Comparative example 2
Large intestine is used in the method and embodiment 1 using Escherichia coli fermentation production poly sialic acid that this comparative example provides
The method that bacillus fermentation produces poly sialic acid is roughly the same, and difference is, the incubation in the step S15 of this comparative example, control
Temperature between cultivation and fermentation 0-48h processed is 40 DEG C, and the temperature after cultivation and fermentation 48h is 40 DEG C.
It samples and is detected during the fermentation tank culture of embodiment 1, comparative example 1 and comparative example 2, specifically, training
Support during, in different time points (fermentation 12h, for 24 hours, 36h, 48h, 60h, 72h) take tunning respectively, detect its OD600
(light absorption value at 600nm wavelength) and poly sialic acid content, as a result as shown in Figure 1, Figure 2 and Figure 3；Wherein poly sialic acid content
Detection method is as follows: by the fermentation liquid centrifugation removal thallus of acquirement, clear liquid dilution, clear liquid and 0.1mol/L after respectively taking 2mL to dilute
Dilute hydrochloric acid mixing, it is closed mixing in 85 DEG C of water-bath 3h, cool down, detected using HPLC after reaction.High performance liquid chromatography
(HPLC) detection method: Shimadzu Lc-15c；Detect column Bio-Rad AMINEX HPX 87H Organic Analysis Column
(300×7.8mm)；60 DEG C of column temperature；Mobile phase 5mmol sulfuric acid, flow velocity are 0.6ml/min；Detection wavelength 210nm.
As shown in Figure 1, in the method for the Escherichia coli fermentation production poly sialic acid that embodiment 1 provides, final PSA yield
For 19.1g/L, synthesis rate 3.18g/L12h, sorb alcohol conversion 10.11%；As shown in Figure 2, comparative example 1 provides
Escherichia coli fermentation produces in the method for poly sialic acid, final PSA yield 15.3g/L, synthesis rate 2.55g/L12h, sorb
Alcohol conversion 8.16%；As shown in Figure 3, in the method for the Escherichia coli fermentation production poly sialic acid that comparative example 2 provides, finally
PSA yield 15.3g/L, synthesis rate 2.55g/L12h, sorb alcohol conversion 8.10%.As it can be seen that using provided by the invention
The cultivation and fermentation technique of interim alternating temperature control can effectively improve PSA fermentation level, combined coefficient and carbon source transformation efficiency,
Promote the fermentation production efficiency of poly sialic acid.
In conclusion the method provided in an embodiment of the present invention for promoting Escherichia coli fermentation production poly sialic acid, Neng Gouyou
Raising carbon source conversion ratio, PSA fermentation level and the PSA combined coefficient of effect, to effectively improve the production efficiency of poly sialic acid.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. a kind of method for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that it is the following steps are included: by large intestine
Bacillus is seeded to fermentation medium cultivation and fermentation, isolated poly sialic acid；When the cultivation and fermentation, cultivation and fermentation 0- is controlled
Temperature between 48h is 40-42 DEG C, and the temperature after control cultivation and fermentation 48h is 36-38 DEG C.
2. the method according to claim 1 for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that the training
When hair care ferment, controlling initial ventilatory capacity is 0.8-1.2vvm, initial speed 150-250rpm；Dissolved oxygen content is adjusted after inoculation
In 3mg/L or more.
3. the method according to claim 1 for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that the training
When hair care ferment, 8h from the Escherichia coli are seeded to the fermentation medium adds ten into the fermentation medium
Six alkyl trimethyl ammonium bromides, make cetyl trimethylammonium bromide reach 150-250mg/L.
4. the method according to claim 1 for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that the training
When hair care ferment, after carbon source has consumed in the fermentation medium, sorbierite is supplemented to the fermentation with the rate of 1-4g/ (Lh)
In culture medium.
5. the method according to claim 1 for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that the training
It the use of ammonia spirit control pH is 6.0-6.5 when hair care ferment.
6. the method according to claim 1 for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that the hair
Ferment culture medium includes following component: the sorbierite of 15-25g/L, the Dried Corn Steep Liquor Powder of 2-4g/L, 3-7g/L dipotassium hydrogen phosphate,
The B race dimension life of the magnesium sulfate of 0.3-0.8g/L, the ammonium chloride of 4-8g/L, the N-acetylglucosamine of 4-8g/L, 0.5-2mL/L
The trace element solution of plain solution and 0.5-2mL/L.
7. the method according to claim 6 for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that the B
Family vitamin solution includes following component: the VB of 0.3-0.6g/L1, 3-3.5g/L VB5, 0.001-0.01g/L VBH and
The VB of 0.1-0.2g/L12。
8. the method according to claim 6 for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that described micro-
Secondary element solution includes following component: the FeSO of 1-4g/L4, 0.01-0.1g/L KIO3, 0.5-1g/L MnCl2、0.1-
The CoCl of 0.5g/L2, 0.01-0.1g/L CrCl3, 0.01-0.2g/L ZnSO4, 0.01-0.05g/L Na2MoO4And 1-
The H of 2g/L3BO3。
9. the method according to claim 6 for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that the hair
Ferment culture medium further includes the sucrose fatty ester of 0.1-1mL/L.
10. the method according to claim 1 for promoting Escherichia coli fermentation production poly sialic acid, which is characterized in that described
The deposit number of Escherichia coli is CCTCC NO:M 2018103.
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| CN201811124478.XA CN109182423B (en) | 2018-09-26 | 2018-09-26 | Method for promoting fermentation of escherichia coli to produce polysialic acid |
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| CN110093293A (en) * | 2019-05-07 | 2019-08-06 | 江苏集萃工业生物技术研究所有限公司 | One plant of Escherichia coli for producing poly sialic acid and its application |
| CN111733092A (en) * | 2020-05-12 | 2020-10-02 | 中科鸿基生物科技有限公司 | Fermentation process of producing polysialic acid and its extracting and refining process |
| CN111733101A (en) * | 2020-06-29 | 2020-10-02 | 嘉必优生物技术（武汉）股份有限公司 | Polysialic acid fermentation medium and method for producing polysialic acid by fermenting escherichia coli |
| CN112553120A (en) * | 2020-12-25 | 2021-03-26 | 华熙生物科技股份有限公司 | Escherichia coli and application thereof in production of polysialic acid |
| CN112608959A (en) * | 2020-12-31 | 2021-04-06 | 河南巨龙生物工程股份有限公司 | Method for improving acetylglucosamine fermentation unit |
| CN117683832A (en) * | 2024-02-04 | 2024-03-12 | 山东润德生物科技有限公司 | Polysialic acid fermentation medium and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093293A (en) * | 2019-05-07 | 2019-08-06 | 江苏集萃工业生物技术研究所有限公司 | One plant of Escherichia coli for producing poly sialic acid and its application |
| CN111733092A (en) * | 2020-05-12 | 2020-10-02 | 中科鸿基生物科技有限公司 | Fermentation process of producing polysialic acid and its extracting and refining process |
| CN111733092B (en) * | 2020-05-12 | 2023-10-31 | 中科鸿基生物科技有限公司 | Method for producing polysialic acid by fermentation and extraction and refining method thereof |
| CN111733101A (en) * | 2020-06-29 | 2020-10-02 | 嘉必优生物技术（武汉）股份有限公司 | Polysialic acid fermentation medium and method for producing polysialic acid by fermenting escherichia coli |
| CN112553120A (en) * | 2020-12-25 | 2021-03-26 | 华熙生物科技股份有限公司 | Escherichia coli and application thereof in production of polysialic acid |
| CN112553120B (en) * | 2020-12-25 | 2021-09-14 | 华熙生物科技股份有限公司 | Escherichia coli and application thereof in production of polysialic acid |
| CN112608959A (en) * | 2020-12-31 | 2021-04-06 | 河南巨龙生物工程股份有限公司 | Method for improving acetylglucosamine fermentation unit |
| CN112608959B (en) * | 2020-12-31 | 2024-04-23 | 河南巨龙生物工程股份有限公司 | Method for improving fermentation unit of acetylglucosamine |
| CN117683832A (en) * | 2024-02-04 | 2024-03-12 | 山东润德生物科技有限公司 | Polysialic acid fermentation medium and application thereof |
| CN117683832B (en) * | 2024-02-04 | 2024-05-14 | 山东润德生物科技有限公司 | Polysialic acid fermentation medium and application thereof |
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