CN106867905A - Preservative fluid and recombination bacillus coli fermentation process in high density for culture presevation - Google Patents
Preservative fluid and recombination bacillus coli fermentation process in high density for culture presevation Download PDFInfo
- Publication number
- CN106867905A CN106867905A CN201510920090.0A CN201510920090A CN106867905A CN 106867905 A CN106867905 A CN 106867905A CN 201510920090 A CN201510920090 A CN 201510920090A CN 106867905 A CN106867905 A CN 106867905A
- Authority
- CN
- China
- Prior art keywords
- culture
- fermentation
- preservative fluid
- high density
- strain
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
This application discloses a kind of preservative fluid for culture presevation and the fermentation process in high density of recombination bacillus coli.The preservative fluid of the application is made up of cushioning liquid with the solute for being dissolved in cushioning liquid, and cushioning liquid is the phosphate buffer or citrate buffer solution of pH value 6.2-7.0, and solute includes oligosaccharide, glycerine, mannitol, skimmed milk power and agarose.The preservative fluid of the application, can make strain long term storage at 0 DEG C -26 DEG C, and keep good vigor, it is to avoid -80 DEG C of traditional preservations are impacted to spawn activity, be that high density fermentation is laid a good foundation.The fermentation process in high density of the recombination bacillus coli of the application, three-class strain preservation is carried out using the preservative fluid of the application, improve the stability of the thalline during long-term culture presevation, effectively maintain spawn activity, greatly reduce production anomaly caused by being degenerated due to spawn activity in fermentation process, meanwhile, be greatly reduced actication of culture wait time, with by with.
Description
Technical field
The application is related to culture presevation field, more particularly to a kind of preservative fluid for culture presevation, and
The fermentation process in high density of the recombination bacillus coli based on the preservative fluid.
Background technology
Recombination bacillus coli, refer to Escherichia coli as host, import recombinant DNA after, being capable of effective expression
One major class genetic engineering bacterium of foreign protein, by the host for being used is Escherichia coli, therefore, it is referred to as
It is recombination bacillus coli.
Current recombination bacillus coli is applied primarily to field of biological pharmacy.Restructuring large intestine is found in process of production
Bacillus stability is not high, in process of production, strain decay easily occurs, and main cause is, 1. traditional
Culture collection process, because freezing in -80 DEG C of refrigerators, it is impossible to the vigor of long-term holding thalline, the work of strain
The problems such as power decline is easily caused thalline self-dissolving, thalline plasmid loss, so as to cause the yield of target metabolic product
Decline；2. during the fermentation, traditional fermentation methods and feed process are unable to reach cell density high so that
The lifting of product yield is restricted.In addition, traditional culture presevation is required for culture presevation at -80 DEG C.
The high density fermentation of recombination bacillus coli is to improve heterogenous expression protein yield and one of quality to have very much
The means of effect, are also the focus and difficult point of current Fermentation Engineering research.Existing research display, influence restructuring is big
The factor of enterobacteria high density fermentation mainly includes, Host Strains exogenous gene expression ability in itself, environment are resistance to
Stress etc., and culture medium, condition of culture, feed process etc..
The content of the invention
The purpose of the application is to provide a kind of preservative fluid for culture presevation and recombination bacillus coli high density hair
Fermenting process, it is intended to effectively keep spawn activity, reducing to be degenerated due to spawn activity in fermentation process is caused
Production anomaly.
To achieve these goals, the application employs following technical scheme：
The one side of the application discloses a kind of preservative fluid for culture presevation, and the preservative fluid is by cushioning liquid
Be dissolved in cushioning liquid solute composition, cushioning liquid for pH value 6.2-7.0 phosphate buffer or
Citrate buffer solution, solute includes oligosaccharide, glycerine, mannitol, skimmed milk power and agarose.
It should be noted that when carrying out preservation to strain using the preservative fluid of the application, can be without -80 DEG C of guarantors
Hide, directly in 0-26 DEG C of preservation, particularly, can be managed for work strain library, but those skilled in the art
Solution, preserving strain using the preservative fluid of the application can also be stored in -80 DEG C.In a kind of preferred side of the application
In case, by work strain library in 4-10 DEG C of preservation, preservation cost is on the one hand reduced, on the other hand, at 4-10 DEG C
Under, it is capable of the holding thalline vigor of longer time, so that for high density fermentation is laid a good foundation, the application's
In a kind of implementation, the thalline survival rate of 538 days is more than 74.8%.
Preferably, oligosaccharide is selected from least one in trehalose, sucrose and maltose.
Preferably, solute includes the oligosaccharide of 100 weight portions, the glycerine of 30-40 weight portions, 50-60 weights
Measure the mannitol of part, the skimmed milk power of 180-200 weight portions, the agarose of 0.5-0.8 weight portions.
Preferably, in preservative fluid, the weight ratio of cushioning liquid and solute is 1:8-15.
The another side of the application discloses the preservative fluid of the application answering in the culture presevation of recombination bacillus coli
With.
The another side of the application also discloses the preservative fluid of the application in the high density fermentation of genetic engineering bacterium
Using.
It should be noted that the preservative fluid of the application be the high density fermentation particular for recombination bacillus coli and
Improvement, it will be understood that the preservative fluid of the application is simultaneously not only limited the use of in recombination bacillus coli；Other genes
Engineering bacteria, can be using the preservative fluid of the application.
The application's simultaneously discloses a kind of fermentation process in high density of recombination bacillus coli again, including uses three
Level culture collection process sets up original strain storehouse, main generation strain library and work strain library, and work strain library is used
The preservative fluid of the application, is stored in -80 DEG C -26 DEG C, is preferably stored in 0-26 DEG C；Also, use work bacterium
The strain in kind of storehouse carries out high density fermentation, high density fermentation sequentially include line culture, shaking table activation culture,
Seed liquor culture, seed tank culture and fermentation tank culture.
It should be noted that in the application, three-class strain method for preserving is specifically included：1. original strain storehouse
Set up：The recombination bacillus coli that will be built cultivates 6-8h, Ran Houyu in being inoculated in 50mL fluid nutrient mediums
The preservative fluid of sterilized equivalent is fully mixed, and is dispensed under aseptic condition into 100 μ L ampoul tubes, less than 0 DEG C
Sealed after Cord blood, or vacuum freeze drying, preserved under normal temperature；2. the foundation of main generation strain library：It is former
Beginning strain library after the assay was approved, takes original strain and shakes pipe activation 6-8h in 10mL nutrient solutions, is then inoculated in
6-8h is cultivated in 50mL fluid nutrient mediums, then is fully mixed with sterilized equivalent preservative fluid, under aseptic condition
In packing to 100 μ L ampoul tubes, sealed after less than 0 DEG C Cord blood, or vacuum freeze drying, under normal temperature
Preserve；3. work the foundation of strain library：Main generation strain library after the assay was approved, takes main generation strain and is cultivated in 10mL
Liquid shakes pipe activation 6-8h, is then inoculated in 50mL fluid nutrient mediums and cultivates 6-8h, then with it is sterilized etc.
The preservative fluid of amount is sufficiently mixed, and is stored in -80 DEG C -26 DEG C.Wherein, the inspection of original strain storehouse and main generation strain library
Inspection can refer to existing inspection and determination methods, do not tire out state herein；In addition, from original strain storehouse
Take bacterium solution to be activated, and bacterium solution is taken from main generation strain library and activated, all referring to prior art, herein
Do not tire out and state.Normal temperature refers to 0-26 DEG C in the application.Work strain library is stored in -80 DEG C to 26 DEG C, preferred to protect
0-26 DEG C is stored in, it is furthermore preferred that being stored in 4 DEG C -10 DEG C.It is appreciated that can using the preservative fluid of the application
Normal temperature is stored in by work strain library, so can conveniently be taken, also, also can more have at normal temperatures
The holding spawn activity of effect, but, equally can be by culture presevation in -80 DEG C using the preservative fluid of the application.
Also, it should be noted that in the application, original strain storehouse and main generation strain library using preservative fluid except being protected
It is stored in beyond normal temperature, it is also possible to which the mass concentration by Liquid Culture product and equivalent is 20% defatted milk mixing
Afterwards, dispense, freeze, be stored in -80 DEG C；Can also mix with the preservative fluid of the application of equivalent, Ran Houbao
It is stored in -80 DEG C.But, in the preferred scheme of the application, original strain storehouse, main generation strain library and work strain
Storehouse is all to be stored in normal temperature using the preservative fluid of the application.
It is appreciated that due to the preservative fluid using the application, preservation of the work strain library at -80 DEG C can be avoided
At a temperature of, cause its vigor to fail, so as to be conducive to the high density fermentation of recombination bacillus coli, as specific
Line culture, shaking table activation culture, seed liquor culture, seed tank culture and fermentation tank culture, Ke Yican
Examining conventional mode is carried out, and does not tire out state herein.Simply, in a kind of preferred implementation of the application, to hair
Fermentation tank culture has carried out special optimization, and density of fermenting is improved with further, and this will be detailed in following scheme
Description.
Preferably, work strain library is stored in 4 DEG C -10 DEG C using the preservative fluid of the application.
It should be noted that using the preservative fluid of the application, culture presevation can be made in 0 DEG C -26 DEG C, but,
In order to reach more preferable preservation effect, it is preferred that be stored in 4 DEG C -10 DEG C.It is appreciated that culture presevation
Effect be, as far as possible reduce or stop strain metabolic activity, its growth and breeding is slowed or stopped；Therefore,
Temperature is lower, more beneficial to reduction or stopping metabolic activity and growth and breeding, but, temperature is too low, such as tradition
- 80 DEG C of preservations, then can influence the vigor of strain, it is particularly long-term to preserve, spawn activity can be made to fail；Cause
This, the application is preferably by the fungi preservation of the strain library that works in 4 DEG C -10 DEG C.
Preferably, fermentation tank culture includes, the bacterium solution of seed tank culture is inoculated into fermentation tank and is ventilated
Stir culture, the initial culture conditions of air agitation culture are, rotating speed 100-150rpm, throughput
0.9-1.2vvm, 35-37 DEG C of temperature, tank pressure 0.03-0.05MPa；When dissolved oxygen is less than 20%, rotating speed is improved
To 200-800rpm, throughput to 1.5-2.5vvm is improved；As fermentation OD600 to more than 30, add
Derivant, meanwhile, fermentation jar temperature is reduced to 25-27 DEG C by 35-37 DEG C, continue to cultivate to fermentation ends；
In whole fermentation process, feed rate is adjusted, pH value keeps 7.0-7.1；Derivant is isopropylthio gala
Glucosides.
Because the beneficial effect using above technical scheme, the application is：
The preservative fluid of the application, can make strain long term storage at 0 DEG C -26 DEG C, and keep good vigor,
Avoid -80 DEG C of traditional preservations from impacting spawn activity, be that high density fermentation is laid a good foundation.The application
Recombination bacillus coli fermentation process in high density, take three-class strain method for preserving, and use the application's
Preservative fluid, substantially increases the stability of the thalline during strain long term storage；Due to culture presevation in
Under 0 DEG C -26 DEG C of temperature conditionss, more effectively maintain spawn activity, greatly reduce in fermentation process due to
Spawn activity produces anomaly caused by degenerating, meanwhile, the time of actication of culture wait has been greatly reduced,
With by with.
Specific embodiment
Existing research display, Host Strains, culture medium, condition of culture, feed process etc. are all that influence restructuring is big
Host Strains, therefore, also there is many research reports, are screened by the key factor of enterobacteria high density fermentation,
Culture medium, condition of culture, feed process etc. are optimized, further to improve the hair of recombination bacillus coli
Ferment density.Present inventor, has found by substantial amounts of research and practice, in addition to factors above, weight
Group Escherichia coli method for preserving also can be direct or indirect influence high density fermentation；The research of the application shows,
Traditional culture collection process is all in -80 DEG C of preservations so that strain cannot maintain vigour for a long time, and strain is lived
Power fails, the problems such as be easily caused thalline self-dissolving, thalline plasmid loss, so as to cause target metabolic product
Yield declines, it is impossible to realize the effect of high density fermentation.But, -80 DEG C of preservations are generally acknowledged culture presevation temperature
Degree, this is almost into the bottleneck that high density fermentation is impassable.
Be based on above research and understanding, the application it is creative propose it is a kind of new for culture presevation
Preservative fluid, the preservative fluid is dissolved in pH value by oligosaccharide, glycerine, mannitol, skimmed milk power and agarose
Formed in 6.6 phosphate buffer or citrate buffer solution.Most of all, the preservative fluid of the application can
So that strain is preserved for a long time at 0-26 DEG C, and keep good vigor, it is to avoid vigor decline is to fermentation
The influence of production.
Preservative fluid based on the application, the application further provides the high density fermentation side of recombination bacillus coli
Method.Although the preservative fluid of the application, its original intention is directed to the high density fermentation of recombination bacillus coli and proposes,
It is appreciated that in order to maintain vigour, any required strain for preserving long-term at 0-26 DEG C can be using this
The preservative fluid of application, the preservative fluid of the application is not limited in recombination bacillus coli and uses, or even also not only limits
Used in high density fermentation.
The application is described in further detail below by specific embodiment.Following examples are only to the application
It is further described, should not be construed as the limitation to the application.
Embodiment
The preservative fluid for culture presevation of this example uses the phosphate buffer of pH6.6 as cushioning liquid,
Solute includes oligosaccharide, glycerine, mannitol, skimmed milk power and agarose, wherein, oligosaccharide is specially sea
Algae sugar, the preparation method of preservative fluid is as follows：
100g oligosaccharide, 30g glycerine, 50g mannitol, 180g skimmed milk powers and 0.5g agaroses are weighed,
Its whole is dissolved in the phosphate buffer of 1000mL50mM.Saved backup after autoclave sterilization sterilization.
The preservative fluid prepared using this example carries out preservation to recombination bacillus coli, then carries out high density fermentation；
The recombination bacillus coli that this example is used is BL21.The fermentation process in high density of this example includes, using three-class strain
Method for preserving sets up original strain storehouse, main generation strain library and work strain library, also, original strain storehouse, master
The preservative fluid that preservation for strain library and work strain library is all prepared using this example, original strain storehouse, main generation bacterium
Plant storehouse and be stored in normal temperature, work strain library is stored in 4 DEG C；Then, height is carried out using the strain of work strain library
Density fermentation, high density fermentation sequentially includes line culture, shaking table activation culture, seed liquor culture, seed
Tank culture and fermentation tank culture.It is specific as follows：
Three-class strain method for preserving：
1. the foundation in original strain storehouse：The recombination bacillus coli that will be built is inoculated in 2% inoculum concentration
6h is cultivated in 50mL fluid nutrient mediums, then the preservative fluid with sterilized equivalent is fully mixed, aseptic condition
In lower packing to 100 μ L ampoul tubes, sealed after vacuum freeze drying, preserved under normal temperature；
2. the foundation of main generation strain library：Original strain storehouse after the assay was approved, 10mL shake pipe activation 6h, then with
2% inoculum concentration cultivates 6h in being inoculated in 50mL fluid nutrient mediums, then abundant with sterilized equivalent preservative fluid
Mix, dispensed under aseptic condition into 100 μ L ampoul tubes, sealed after vacuum freeze drying, preserved under normal temperature；
3. work the foundation of strain library：Main generation strain library after the assay was approved, 10mL shake pipe activation 6h, then with
2% inoculum concentration cultivates 6h in being inoculated in 50mL fluid nutrient mediums, then with the preservation liquid of sterilized equivalent
It is sufficiently mixed, is stored in 4 DEG C.
It should be noted that the incubation time that pipe activation is cultivated or shaken in this example, in fluid nutrient medium is 6-8h
.As for the storage temperature of work strain library, at 0-26 DEG C, 4 DEG C of -10 DEG C of energy are preferably stored in
The holding spawn activity of enough longer times.Preserved under the normal temperature of this example, its temperature is 0-26 DEG C, preferred to protect
It is stored in 4 DEG C -10 DEG C.
In the three-class strain method for preserving of this example, fluid nutrient medium is LB culture mediums, shakes pipe activation and is used
Culture medium be also LB culture mediums.
In the three-class strain method for preserving of this example, original strain storehouse and main generation strain library are except the guarantor using this example
Liquid is hidden to be stored in beyond normal temperature, can also be by Liquid Culture product and the degreasing that the mass concentration of equivalent is 20%
After milk is mixed, packing, lyophilized -80 DEG C are stored in；Or the same preservative fluid using this example, by Liquid Culture
After product is mixed with the preservative fluid of equivalent, it is stored under less than 0 DEG C of cryogenic conditions.
After establishing three-class strain storehouse, original strain storehouse and main generation strain library continue to save backup, and take work bacterium
The strain in kind of storehouse carries out high density fermentation, sequentially including line culture, shaking table activation culture, seed liquor culture,
Seed tank culture and fermentation tank culture.It is specific as follows：
Line culture：Strain in the strain library that works is carried out into line culture 24h, inclined-plane in slant medium
The component of culture medium includes parts by weight：Peptone 1.5%, extraction from yeast powder 1%, NaCl 1%, agar powder
2.0%, balance of deionized water, Medium's PH Value is 6.8-7.0, and this example is specially pH 7.0；
Shaking table activation culture：The thalline of picking line culture, is inoculated in shaking table activation in 50mL fluid nutrient mediums
Culture 8h, the fluid nutrient medium that this example is used is LB culture mediums；
Seed liquor culture：By the strain transfer after shaking table activation culture in 500mL, inoculum concentration 1%, liquid training
Shaking table culture 8h in base is supported, seed liquor is obtained, the nutrient solution that seed liquor culture is used includes parts by weight：
Peptone 1.5%, extraction from yeast powder 1%, NaCl 1%, balance of deionized water, medium pH value is 6.8-7.0
, this example is specially pH 7.0；
Seed tank culture：The seed liquor of seed liquor culture is inoculated in sterilized seeding tank with 2% ratio
In, air agitation culture 6h, condition of culture is：Rotating speed 300rpm, throughput 1vvm, 35-37 DEG C of temperature
, this example is specially 37 DEG C, tank pressure 0.05MPa；The nutrient solution that seed tank culture is used includes weight
Number：Peptone 1.5%, extraction from yeast powder 2.0%, NaCl 1%, glucose 2%, KH2PO40.1%th,
K2HPO40.2%, balance of deionized water, medium pH value is 6.8-7.0, and this example is specially pH 7.0；
Fermentation tank culture：The bacterium solution of seed tank culture is inoculated in sterilized fermentation tank with 2% ratio
Row air agitation culture, the initial culture conditions of air agitation culture are, rotating speed 120rpm, throughput 1.0vvm,
35-37 DEG C of culture, this example is specially 37 DEG C, tank pressure 0.05MPa；When dissolved oxygen is less than 20%, improve
Rotating speed improves throughput to 2.0vvm to 400rpm；As fermentation OD600 to more than 30, induction is added
Agent, meanwhile, fermentation jar temperature is reduced to 25-27 DEG C by 37 DEG C, this example will be specifically 27 DEG C, after
Continuous culture 10-16h, this example is specially 16h, fermentation ends.In whole fermentation process, feed rate is adjusted,
Control pH7.0-7.1, it is 7.0 specifically to control pH, and the derivant that this example is used is isopropylthio galactolipin
Glycosides.In this example, the nutrient solution that fermentation tank culture is used includes parts by weight：Peptone 2%, extraction from yeast powder
2.0%th, NaCl 1%, glucose 1%, KH2PO40.20%th, K2HPO40.35%th, MgSO40.05%,
Balance of deionized water, medium pH value is 6.8-7.0, and this example is specially pH 7.0.Feed supplement is adopted
Nutrient solution includes parts by weight：Peptone 3%, extraction from yeast powder 8%, glycerine 10%, ZnSO40.6%th,
CaCl20.5%th, CoCl20.3%th, MgSO43%, balance of deionized water, medium pH value is 6.8-7.5
, this example is specially pH 7.0.
After fermentation ends, the fermentation density of this example tunning is measured, as a result shown, this example restructuring large intestine bar
The fermentation density of bacterium is OD140；Fermentation density through measuring dry cell weight is 50g/L.It should be noted that
Even if the fermentation density of dry cell weight is generally in more than 30g/L high density fermentation bacterium, and the application can reach
50g/L, exceeds well over common standard.
In addition, the survival rate of the strain of the work strain library set up to this example three-class strain method for preserving is carried out
Detection.Result shows that work strain library preservation 332 days under the conditions of 4 to 10 DEG C, strain survival rate is 81.2%
More than, the survival rate of preservation 538 days is more than 74.8%.The strain survival rate of preservation 10 days exists at 25 DEG C
More than 70.9%, the survival rate of preservation 20 weeks is more than 51.8%.
It can be seen that, the work strain library of this example can be preserved for a long time under the conditions of 4 to 10 DEG C, and can be compared with
Good holding spawn activity, compared to -80 DEG C of traditional preservations, in addition it is also necessary to carry out defrosting, recovery of strain etc.
A little row work, it is more convenient and practical.
On the basis of being tested more than, this example further to different cushioning liquid, different oligosaccharide, with
And the consumption of preservative fluid each component is tested.Result shows, cushioning liquid except phosphate buffer with
Outward, can also be using the citrate buffer solution of pH value 6.6；Oligosaccharide can also use sucrose or maltose,
Replace trehalose；The consumption of preservative fluid each component, according to the 100 parts of calculations of oligosaccharide weight portion, the weight of glycerine
Part 30-40, the weight portion 50-60 of mannitol, the weight portion 180-200 of skimmed milk power, the weight portion of agarose
0.5-0.8, can prepare the preservative fluid suitable with this example effect.
Above content is to combine the further description that specific embodiment is made to the application, it is impossible to recognized
The specific implementation for determining the application is confined to these explanations.For the ordinary skill of the application art
For personnel, on the premise of the application design is not departed from, some simple deduction or replace can also be made,
The protection domain of the application should be all considered as belonging to.
Claims (10)
1. a kind of preservative fluid for culture presevation, it is characterised in that：By cushioning liquid and being dissolved in cushioning liquid
In solute composition, the cushioning liquid for pH value 6.2-7.0 phosphate buffer or citrate buffer solution,
The solute includes oligosaccharide, glycerine, mannitol, skimmed milk power and agarose.
2. preservative fluid according to claim 1, it is characterised in that：The oligosaccharide is selected from trehalose, sugarcane
At least one in sugar and maltose.
3. preservative fluid according to claim 1 and 2, it is characterised in that：The solute includes 100 weights
The oligosaccharide of amount part, the glycerine of 30-40 weight portions, the mannitol of 50-60 weight portions, 180-200 weight portions
Skimmed milk power, the agarose of 0.5-0.8 weight portions.
4. preservative fluid according to claim 1 and 2, it is characterised in that：It is described in the preservative fluid
The weight ratio of cushioning liquid and the solute is 1:(8-15).
5. a kind of preservative fluid as described in claim any one of 1-4 is in the culture presevation of recombination bacillus coli
Using.
6. a kind of preservative fluid as described in claim any one of 1-4 is in the high density fermentation of genetic engineering bacterium
Application.
7. a kind of fermentation process in high density of recombination bacillus coli, it is characterised in that：Including being protected using three-class strain
Tibetan method sets up original strain storehouse, main generation strain library and work strain library, and the work strain library uses right
It is required that the preservative fluid described in any one of 1-4, is stored in -80 DEG C to 26 DEG C, 0 DEG C -26 DEG C are preferably stored in；
Also, high density fermentation is carried out using the strain of the work strain library, the high density fermentation sequentially includes
Line culture, shaking table activation culture, seed liquor culture, seed tank culture and fermentation tank culture.
8. fermentation process in high density according to claim 7, it is characterised in that：The work strain library is adopted
With the preservative fluid described in claim any one of 1-4,4 DEG C -10 DEG C are stored in.
9. fermentation process in high density according to claim 7, it is characterised in that：The original strain storehouse and
/ or main generation strain library be also, using the preservative fluid described in claim any one of 1-4, to be stored in -80 DEG C to 26 DEG C,
Preferably it is stored in 0 DEG C -26 DEG C.
10. the fermentation process in high density according to claim any one of 7-9, it is characterised in that：The hair
Fermentation tank culture includes that the bacterium solution of the seed tank culture is inoculated into fermentation tank carries out air agitation culture,
The initial culture conditions of the air agitation culture are, rotating speed 100-150rpm, throughput 0.9-1.2vvm,
35-37 DEG C of temperature, tank pressure 0.03-0.05MPa；When dissolved oxygen is less than 20%, rotating speed is improved to 200-800rpm,
Improve throughput to 1.5-2.5vvm；As fermentation OD600 to more than 30, derivant is added, meanwhile, will
Fermentation jar temperature is reduced to 25 DEG C -27 DEG C by 35 DEG C -37 DEG C, continues to cultivate to fermentation ends；Whole fermentation process
In, feed rate is adjusted, pH value is kept 7.0-7.1；The derivant is isopropylthiogalactoside.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510920090.0A CN106867905A (en) | 2015-12-11 | 2015-12-11 | Preservative fluid and recombination bacillus coli fermentation process in high density for culture presevation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510920090.0A CN106867905A (en) | 2015-12-11 | 2015-12-11 | Preservative fluid and recombination bacillus coli fermentation process in high density for culture presevation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106867905A true CN106867905A (en) | 2017-06-20 |
Family
ID=59178566
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510920090.0A Pending CN106867905A (en) | 2015-12-11 | 2015-12-11 | Preservative fluid and recombination bacillus coli fermentation process in high density for culture presevation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106867905A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292752A (en) * | 2021-12-31 | 2022-04-08 | 山东新时代药业有限公司 | Freeze-dried composition of recombinant escherichia coli strain and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1358838A (en) * | 2002-01-18 | 2002-07-17 | 南京农业大学 | Preservation method for live bacterial preparation |
| WO2009020455A1 (en) * | 2007-08-08 | 2009-02-12 | Conjugon, Inc. | Compositions and methods for microbe storage and delivery |
| CN102643749A (en) * | 2012-04-28 | 2012-08-22 | 中粮生物化学（安徽）股份有限公司 | Microorganism low-temperature preservation protective agent and microorganism low-temperature preservation method |
| CN104293668A (en) * | 2013-07-17 | 2015-01-21 | 南京朗恩生物科技有限公司 | Recombinant escherichia coli high-density fermentation method |
-
2015
- 2015-12-11 CN CN201510920090.0A patent/CN106867905A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1358838A (en) * | 2002-01-18 | 2002-07-17 | 南京农业大学 | Preservation method for live bacterial preparation |
| WO2009020455A1 (en) * | 2007-08-08 | 2009-02-12 | Conjugon, Inc. | Compositions and methods for microbe storage and delivery |
| CN102643749A (en) * | 2012-04-28 | 2012-08-22 | 中粮生物化学（安徽）股份有限公司 | Microorganism low-temperature preservation protective agent and microorganism low-temperature preservation method |
| CN104293668A (en) * | 2013-07-17 | 2015-01-21 | 南京朗恩生物科技有限公司 | Recombinant escherichia coli high-density fermentation method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292752A (en) * | 2021-12-31 | 2022-04-08 | 山东新时代药业有限公司 | Freeze-dried composition of recombinant escherichia coli strain and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Production of chitinase from Verticillium lecanii F091 using submerged fermentation | |
| CN107287121A (en) | A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof and application method | |
| CN107034149A (en) | A kind of kluyveromyces marxianus bacterium freezes the preparation method and its usage of microbial inoculum | |
| CN108739057B (en) | Method for prolonging storage time of solid strain of edible fungus | |
| CN102757917B (en) | Biocontrol agent for preventing and curing bacterial wilt and club roots of plant as well as preparation method and application thereof | |
| Narta et al. | Improved production of L-asparaginase by Bacillus brevis cultivated in the presence of oxygen-vectors | |
| Li et al. | Combined effects of endo-and exogenous trehalose on stress tolerance and biocontrol efficacy of two antagonistic yeasts | |
| Diaz et al. | Simultaneous saccharification and fermentation of industrial sweetpotatoes for ethanol production and anthocyanins extraction | |
| CN106520802A (en) | GAD gene for increasing stress tolerance of lactic acid bacteria and application thereof | |
| CN107043797A (en) | The technique that a kind of fermentation by saccharomyces cerevisiae produces glutathione | |
| CN111235039B (en) | Culture medium for culturing metarhizium anisopliae with high toxicity, preparation and application thereof | |
| Zhang et al. | Burdock fructooligosaccharide enhances biocontrol of Rhodotorula mucilaginosa to postharvest decay of peaches | |
| CN105062891A (en) | Enzyme-containing composition and method for enhancing stability of liquid microbial inoculant | |
| CN110713934A (en) | Edible fungus production strain preservation method and activation method thereof | |
| CN100352914C (en) | Vacuum freeze-dried product of antagonist bacteria C.laurentii of yeast and its preparation method | |
| CN106867905A (en) | Preservative fluid and recombination bacillus coli fermentation process in high density for culture presevation | |
| CN101818183B (en) | Method for improving trehalose content of yeast cell | |
| CN105925495A (en) | Highly-active Pichia pastoris powder and preparation method thereof | |
| CN105821092A (en) | Hainanmycin fermentation process | |
| CN103421693B (en) | The method of rihizobium japonicum vitrification frozen stock solution and glass frozen preservation rihizobium japonicum | |
| CN109294916A (en) | A kind of culture presevation agent and the method using the preserving agent anaerobism freezing genetic engineering bacterium | |
| CN108977379A (en) | A kind of microorganism low-temperature preservation protective agent | |
| CN109294917A (en) | A kind of culture presevation agent and the method using the preserving agent superfreeze preservation genetic engineering bacterium | |
| CN110122561A (en) | Olive Candida promotes to adopt the callus method of rear potato tubers | |
| CN102399803B (en) | Improved beta-glucosidase gene and preparation of recombinase thereof |
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 | ||
| CB02 | Change of applicant information |
Address after: Beishan Industrial Zone Building in Yantian District of Shenzhen city of Guangdong Province in 518083 Applicant after: BGI SHENZHEN Address before: Beishan Industrial Zone Building in Yantian District of Shenzhen city of Guangdong Province in 518083 Applicant before: BGI SHENZHEN |
|
| CB02 | Change of applicant information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180111 Address after: 518083 comprehensive building of Beishan industrial zone and 11 Building 2, Yantian District, Guangdong, Shenzhen Applicant after: MGI TECH Co.,Ltd. Address before: Beishan Industrial Zone Building in Yantian District of Shenzhen city of Guangdong Province in 518083 Applicant before: BGI SHENZHEN |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170620 |
|
| RJ01 | Rejection of invention patent application after publication |