CN109082388A - E. coli isolated from ducks high density fermentation culture medium and preparation method thereof - Google Patents
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Abstract
The invention discloses a kind of E. coli isolated from ducks high density fermentation culture mediums and preparation method thereof, the culture medium prescription is to contain 30.0~40.0g of yeast extract in every liter of water, 5.0~15.0g of tryptone, 5.0~15.0g of soy peptone, 5.0~10.0g of lactoalbumin hydrolysate, 5.0~10.0g of casein peptone, 4.0~7.0g of stomach cardia peptone, 1.0~3.0g of fructus hordei germinatus leaching powder, 2.0~5.0g of dipotassium hydrogen phosphate, 1.0~3.0mL of glycerol, 2.0~10.0mg of molysite, 2.0~10.0mg of manganese salt, 2.0~10.0mg of aluminium salt.Culture medium of the present invention is cheap, and preparation method is simple, it can be achieved that E. coli isolated from ducks high density fermentation, can be used for large scale fermentation production E. coli isolated from ducks vaccine.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to a kind of E. coli isolated from ducks high density fermentation culture medium and
Preparation method.
Background technique
Duck E. coli is caused by enteropathogenic E. Coli, and pathological manifestations mainly have acute sepsis, air bag inflammation, full eye
Ball inflammation, arthrosynovitis, salpingitis and peritonitis etc..With the continuous expansion of cultivation scale, aquatic bird raises water environment
Constantly deteriorate, colibacillosis has become to endanger aquatic bird and cultivate more serious one of bacterial infectious disease at present.Due to facing at present
The bacteriosis such as antibiotic treatment Escherichia coli are widely used on bed, lead to the generation of a large amount of antibody-resistant bacterium, therefore, in large intestine
It the use of vaccine immunization is effective prevention approach in bacillus prevention and treatment.
For bacterial vaccine, how to improve bacterium bacterium number (i.e. antigenic content) be bacterial vaccine research key technology it
One.In general, after the high vaccine organism of antigenic content, higher antibody level can be generated, the antibody duration also compared with
It is long.Bacterium high-density fermentation technology had become common culture technique means, the technology in bacterial vaccine preparation already can be improved list
The concentration of thallus in the volume culture solution of position, and then improve volume productivity.
Enteropathogenic E. Coli is facultative anaerobic bacteria, of less demanding to condition of culture, in common LB broth bouillon
Energy normal growth, however it is difficult to realize its high density fermentation, pathogenic E. coli isolated from ducks high density fermentation is even more to be rarely reported.
Summary of the invention
It is an object of that present invention to provide a kind of E. coli isolated from ducks novel high-density fermentation mediums and preparation method thereof.Purpose
It is
The technical solution used in the present invention is:
The formula of E. coli isolated from ducks high density fermentation culture medium, the culture medium is to contain yeast extract in every liter of water
30.0~40.0g, 5.0~15.0g of tryptone, 5.0~15.0g of soy peptone, 5.0~10.0g of lactoalbumin hydrolysate, junket egg
White 5.0~10.0g of peptone, stomach cardia 4.0~7.0g of peptone, 1.0~3.0g of fructus hordei germinatus leaching powder, 2.0~5.0g of dipotassium hydrogen phosphate, glycerol
1.0~3.0mL, 2.0~10.0mg of molysite, 2.0~10.0mg of manganese salt, 2.0~10.0mg of aluminium salt.
Further, the molysite is selected from least one of ferric nitrate, iron chloride, ferric sulfate.
Further, the manganese salt is selected from least one of manganese sulfate, manganese chloride.
Further, the aluminium salt is selected from least one of aluminum sulfate, aluminium chloride.
Further, the formula of the culture medium is to contain yeast extract 40.0g, tryptone 10.0g in every liter of water,
Soy peptone 10.0g, lactoalbumin hydrolysate 5.0g, casein peptone 10.0g, stomach cardia peptone 5.0g, fructus hordei germinatus leaching powder 2.0g, phosphoric acid hydrogen
Dipotassium 5.0g, glycerol 2.0mL, ferric nitrate 5.0mg, manganese sulfate 5.0mg, aluminum sulfate 5.0mg.
Application of the E. coli isolated from ducks high density fermentation culture medium described in any of the above embodiments in E. coli isolated from ducks fermentation.
E. coli isolated from ducks high density fermentation culture medium described in any of the above embodiments is preparing answering in E. coli isolated from ducks vaccine
With.
The preparation method of E. coli isolated from ducks high density fermentation culture medium, comprising the following steps: yeast leaching is added in every liter of water
30.0~40.0g of object out, 5.0~15.0g of tryptone, 5.0~15.0g of soy peptone, 5.0~10.0g of lactoalbumin hydrolysate,
5.0~10.0g of casein peptone, stomach cardia 4.0~7.0g of peptone, 1.0~3.0g of fructus hordei germinatus leaching powder, dipotassium hydrogen phosphate 2.0~5.0g are sweet
Oil 1.0~3.0mL, 2.0~10.0mg of molysite, 2.0~10.0mg of manganese salt, 2.0~10.0mg of aluminium salt are dissolved by heating, 120~
130 DEG C of 10~20min of high pressure sterilization.
The beneficial effects of the present invention are:
E. coli isolated from ducks high density fermentation culture medium of the present invention is formed using special component, can be used for pathogenic duck large intestine bar
E. coli isolated from ducks vaccine is mass produced in bacterium high density fermentation.E. coli isolated from ducks high density fermentation culture medium of the invention can have
Effect improves thalli growth concentration and the speed of growth in unit volume culture solution, and then improves volume productivity, reduces production cost.
Detailed description of the invention
Fig. 1 is the thallus Gram's staining carried out after E. coli isolated from ducks high density fermentation using different culture medium.
Growth curve of Fig. 2 E. coli isolated from ducks in high density fermentation culture medium of the present invention, LB broth bouillon.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
The present invention is not particularly limited the source of E. coli isolated from ducks and serotype, in the specific embodiment of the invention,
The E. coli isolated from ducks used separates the O78 serotype E. coli isolated from ducks velogen strain identified and saved for this laboratory.Duck large intestine bar
The upgrowth situation of bacterium is different from existing laboratory engineering bacteria (such as recombination bacillus coli), and existing culture medium is difficult to realize pathogenic duck
The high density fermentation of Escherichia coli.
A kind of E. coli isolated from ducks high density fermentation culture medium of embodiment 1
The formula of the present embodiment E. coli isolated from ducks high density fermentation culture medium are as follows: yeast extract 40.0g, tryptone
10.0g, soy peptone 10.0g, lactoalbumin hydrolysate 5.0g, casein peptone 10.0g, stomach cardia peptone 5.0g, fructus hordei germinatus leaching powder 2.0g,
Dipotassium hydrogen phosphate 5.0g, glycerol 2.0mL, ferric nitrate 5.0mg, manganese sulfate 5.0mg, aluminum sulfate 5.0mg heat above-mentioned each component
Constant volume is dissolved in 1L water, 121 DEG C of high pressure sterilization 15min.
Comparative example 1LB broth bouillon
The preparation method of LB broth bouillon are as follows: weigh yeast extract 5.0g, tryptone 10.0g, sodium chloride
10.0g, by above-mentioned each component dissolution constant volume in 1L water, 121 DEG C of high pressure sterilization 15min；To obtain the final product.
Comparative example 2
Soy peptone in the formula of embodiment 1 is substituted for glutin peptone, other compositions and the same embodiment of dosage
1。
Comparative example 3
Lactoalbumin hydrolysate in the formula of embodiment 1 is substituted for glutin peptone, other compositions and the same embodiment of dosage
1。
Comparative example 4
Casein peptone in the formula of embodiment 1 is substituted for glutin peptone, other compositions and dosage are the same as embodiment 1.
Comparative example 5
Stomach cardia peptone in the formula of embodiment 1 is substituted for glutin peptone, other compositions and dosage are the same as embodiment 1.
Comparative example 6
In addition to " high density fermentation culture medium " in embodiment 1 to be substituted for, " high density containing 1 (w/v) % glucose is sent out
Ferment culture medium ", other operations are the same as embodiment 1." high density fermentation culture medium for containing 1 (w/v) % glucose " and " high density hair
Unique difference of ferment culture medium " is exactly that joined glucose in high density fermentation culture medium, and wherein glucose is in the medium
Concentration be 1 (w/v) %.
Comparative example 7
Fructus hordei germinatus leaching powder in the formula of embodiment 1 is substituted for maltose, other compositions and dosage are the same as embodiment 1.
Comparative example 8
Dipotassium hydrogen phosphate in the formula of embodiment 1 is substituted for potassium dihydrogen phosphate, other compositions and the same embodiment of dosage
1。
Comparative example 9
Dipotassium hydrogen phosphate in the formula of embodiment 1 is substituted for sodium chloride, other compositions and dosage are the same as embodiment 1.
Comparative example 10
Ferric nitrate in the formula of embodiment 1 is substituted for calcium chloride, other compositions and dosage are the same as embodiment 1.
Comparative example 11
Manganese sulfate in the formula of embodiment 1 is substituted for calcium chloride, other compositions and dosage are the same as embodiment 1.
Comparative example 12
Aluminum sulfate in the formula of embodiment 1 is substituted for calcium chloride, other compositions and dosage are the same as embodiment 1
A kind of E. coli isolated from ducks high density fermentation culture medium of embodiment 2
The formula of the present embodiment E. coli isolated from ducks high density fermentation culture medium are as follows: yeast extract 30.0, tryptone
5.0g, soy peptone 15.0g, lactoalbumin hydrolysate 10.0g, casein peptone 5.0g, stomach cardia peptone 4.0g, fructus hordei germinatus leaching powder 3.0g,
Dipotassium hydrogen phosphate 2.0g, glycerol 1.0mL, ferric nitrate 10.0mg, manganese sulfate 2.0mg, aluminum sulfate 10.0mg, water 1L.In 121 DEG C of height
Pressure sterilizing 15min；To obtain the final product.
A kind of E. coli isolated from ducks high density fermentation culture medium of embodiment 3
The formula of the present embodiment E. coli isolated from ducks high density fermentation culture medium are as follows: yeast extract 35.0g, tryptone
15.0g, soy peptone 5.0g, lactoalbumin hydrolysate 8.0g, casein peptone 8.0g, stomach cardia peptone 7.0g, fructus hordei germinatus leaching powder 1.0g, phosphorus
Sour hydrogen dipotassium 3.0g, glycerol 3.0mL, ferric nitrate 2.0mg, manganese sulfate 10.0mg, aluminum sulfate 2.0mg, water 1L.In 121 DEG C of high pressures
Sterilize 15min；To obtain the final product.
Culture medium in above-described embodiment 1~3, comparative example 1~12 is subjected to bacterium (O78 with small sample 100mL conical flask
Serotype E. coli isolated from ducks) inoculated and cultured, 3 repetitions of every group of setting, 37 DEG C of shaking table cultures, respectively 16h, 18h, 20h sample,
OD600 is measured using ultraviolet specrophotometernmAbsorbance value.Testing result is as shown in table 1.
1 different culture medium of table is to O78 serotype E. coli isolated from ducks culture situation
As can be seen that screened nitrogen source ingredient, E. coli isolated from ducks can be leached using yeast from the testing result in table 1
Object, tryptone, soy peptone, lactoalbumin hydrolysate, casein peptone and stomach cardia peptone, however glutin cannot be utilized
Peptone；Screened carbon source ingredient, E. coli isolated from ducks can utilize glycerol and fructus hordei germinatus leaching powder, however glucose and maltose obviously inhibit
Growth, is analyzed according to later data, adds glucose that can be obviously promoted the proliferation of Escherichia coli in ferment middle stream, anti-with this side
It reflects, Escherichia coli Growth adds glucose in earlier fermentation, and metabolite will inhibit the growth of Escherichia coli early period.It is screened
Inorganic salts ingredients analysis, dipotassium hydrogen phosphate can promote the growth of Escherichia coli, and sodium chloride and potassium dihydrogen phosphate are then light wherein
Micro- inhibition E. coli isolated from ducks growth；Screened microelement, three kinds of iron, manganese and aluminium microelements can promote Escherichia coli Growth
Fermentation, however do not utilize microelements of calcium.The above results explanation, the specific high density fermentation culture medium of the present invention are very beneficial for duck
The growth of Escherichia coli is fermented, and the effect of other formula culture mediums is significantly better than.
A kind of method that high density fermentation culture is carried out to E. coli isolated from ducks using high density fermentation culture medium of embodiment 4
Firstly, preparing preferred E. coli isolated from ducks high density fermentation culture medium and related reagent, going out for fermentor is carried out
Tank processing and assembling:
A. high density fermentation culture medium: with embodiment 1,1L volume is prepared.
B.2M sulfuric acid solution: 98% concentrated sulfuric acid 109mL is measured, is slowly added in 800mL water, after cooling plus water is settled to
1L volume is transferred in fermentor acid pump feed supplement bottle, 121 DEG C of high pressure sterilization 15min.
C.6M ammonia spirit: pure ammonia spirit 230mL is measured, water is added to be settled to 1L, 0.22um filter filtration sterilization is sterile
Operation is transferred to after sterilizing in alkali pump feed supplement bottle.
D.1 (v/v) %204 defoams agent solution: measuring 204 defoaming agent of 1mL, constant volume is transferred to fermentor defoaming in 1L water
It pumps in feed supplement bottle, 121 DEG C of high pressure sterilization 15min.
E.50 (w/v) % glucose solution: weighing glucose 500g, dissolves by heating constant volume in 1L water, 0.22um filter
Filtration sterilization, sterile working, glucose feeding pumps in feed supplement bottle after being transferred to sterilizing.
Secondly, the preparation of fermentation seed liquid, as previously mentioned, it is strong to take out O78 serotype E. coli isolated from ducks from -80 DEG C of refrigerators
Strain, streak inoculation is placed in 37 DEG C of incubator cultures 12~16 hours on LB plain agar culture medium after defrosting.Then, make
With E. coli isolated from ducks monoclonal colonies on oese picking LB plain agar plate, it is inoculated in 5mL high density fermentation culture medium,
37 DEG C of shaking tables are cultivated 12~16 hours under the conditions of 180rpm.In 1:100 ratio, the monoclonal bacterium solution for taking 1mL to cultivate connects respectively again
Kind is in 100mL high density fermentation culture medium, 37 DEG C of shaking tables, cultivates under the conditions of 180rpm to logarithmic growth phase, the seed of optimization
Bacterium initial concentration OD600Nm is 1.0, in this, as fermentation kind of a daughter bacteria.
Fermentor goes out after tank, and assembly and connection finishes, and adds 1 (v/v) %204 to defoam agent solution 20 by peristaltic pump stream before inoculation
~50mL takes the fermentation of above-mentioned preparation to be seeded in the ratio of 1:10 containing preferred novel high-density fermentation medium with kind of daughter bacteria
Fermentor in ferment, be arranged pH7.0,37 DEG C of temperature.
Earlier fermentation, revolving speed setting are stepped up by 100rpm to 400rpm, and air vent amount is gradually mentioned by 1.5L/min
Rising to 5L/min, (point raises speed between timesharing, fermentation liquid OD600Nm reaches 5.0~8.0 and takes around 3-4 hours, when just having connect bacterium
100rpm is arranged in revolving speed, and 150rpm is arranged in ventilatory capacity 1.5L/min, 0.5h, and 200rpm is arranged in 2L/min, 1h, and 3L/min, 2h are set
300rpm, 4L/min are set, 3h is set as 400rpm, 5L/min.), by control air vent amount and revolving speed, make fermentation liquid
OD600Nm is rapidly achieved between 4.0~6.0.Entire fermentation process pH is molten by acid and alkali pumps auto-feeding 6M ammonia spirit and 2M sulfuric acid
Liquid is controlled, and entire E. coli isolated from ducks fermentation process produces acid, and the consumption of ammonium hydroxide is more.
As fermentation liquid OD600When nm reaches 5.0 or so, is flowed at a slow speed by constant speed feed supplement pump with flow velocity 15mL/h/L and add 50 (w/
V) % glucose solution 200mL.Gravity flow plus glucose start, fixed air vent amount 5L/min, revolving speed 400rpm.
As the OD of fermentation liquid600When nm value stops increasing, fermentation ends.
Sampling in every 2 hours is primary during entire fermentation, measures OD600Nm value draws growth curve.Logarithmic growth phase
Bacterium solution carries out Gram's staining and observes ne ar.
Comparative example 13
In addition to the high density fermentation culture medium in embodiment 4 is substituted for LB broth bouillon, (specific formula is shown in comparative example
1) outside, other all operations are the same as embodiment 4.
Compare E. coli isolated from ducks in high density fermentation culture medium, the growth curve and thalli morphology of LB broth bouillon.
As a result:
Gram's staining result is feminine gender as shown in Figure 1, Gram's staining, short corynebacteria, and no significant difference is said
Bright E. coli isolated from ducks can be with normal growth in high density fermentation culture medium of the present invention.
Compare growth curve of the E. coli isolated from ducks in high density fermentation culture medium, LB meat soup culture, as shown in Fig. 2, duck
The speed of growth of the Escherichia coli in novel high-density fermentation medium is obviously faster than LB broth bouillon, bacterium stationary phase thallus
Concentration (OD600Nm value is 30.51) to be also significantly greater than LB broth bouillon (OD60013.78) nm value is.
The above results illustrate that the speed of growth and growth concentration of the E. coli isolated from ducks in high density fermentation culture medium are high
In LB broth bouillon, it was demonstrated that high density fermentation culture medium of the present invention is particularly important for the high density fermentation of E. coli isolated from ducks can
As E. coli isolated from ducks vaccine large-scale production culture medium.It is extensive that culture medium of the present invention can be used for pathogenic E. coli isolated from ducks
High density fermentation can effectively improve volume productivity, reduce production cost.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (8)
1. E. coli isolated from ducks high density fermentation culture medium, which is characterized in that the formula of the culture medium is to contain yeast in every liter of water
30.0~40.0g of extract, 5.0~15.0g of tryptone, 5.0~15.0g of soy peptone, lactoalbumin hydrolysate 5.0~
10.0g, 5.0~10.0g of casein peptone, stomach cardia 4.0~7.0g of peptone, 1.0~3.0g of fructus hordei germinatus leaching powder, dipotassium hydrogen phosphate 2.0~
5.0g, 1.0~3.0mL of glycerol, 2.0~10.0mg of molysite, 2.0~10.0mg of manganese salt, 2.0~10.0mg of aluminium salt.
2. E. coli isolated from ducks high density fermentation culture medium according to claim 1, which is characterized in that the molysite is selected from nitre
At least one of sour iron, iron chloride, ferric sulfate.
3. E. coli isolated from ducks high density fermentation culture medium according to claim 1, which is characterized in that the manganese salt is selected from sulphur
At least one of sour manganese, manganese chloride.
4. E. coli isolated from ducks high density fermentation culture medium according to claim 1, which is characterized in that the aluminium salt is selected from sulphur
At least one of sour aluminium, aluminium chloride.
5. E. coli isolated from ducks high density fermentation culture medium according to claim 1, which is characterized in that the formula of the culture medium
For, yeast extract 40.0g, tryptone 10.0g, soy peptone 10.0g, lactoalbumin hydrolysate 5.0g are contained in every liter of water,
Casein peptone 10.0g, stomach cardia peptone 5.0g, fructus hordei germinatus leaching powder 2.0g, dipotassium hydrogen phosphate 5.0g, glycerol 2.0mL, ferric nitrate 5.0mg,
Manganese sulfate 5.0mg, aluminum sulfate 5.0mg.
6. the described in any item E. coli isolated from ducks high density fermentation culture mediums of Claims 1 to 5 are in E. coli isolated from ducks fermentation
Using.
7. the described in any item E. coli isolated from ducks high density fermentation culture mediums of Claims 1 to 5 are preparing E. coli isolated from ducks vaccine
In application.
8. the preparation method of E. coli isolated from ducks high density fermentation culture medium, which comprises the following steps: add in every liter of water
Enter 30.0~40.0g of yeast extract, 5.0~15.0g of tryptone, 5.0~15.0g of soy peptone, lactoalbumin hydrolysate 5.0
~10.0g, 5.0~10.0g of casein peptone, stomach cardia 4.0~7.0g of peptone, 1.0~3.0g of fructus hordei germinatus leaching powder, dipotassium hydrogen phosphate 2.0
~5.0g, 1.0~3.0mL of glycerol, 2.0~10.0mg of molysite, 2.0~10.0mg of manganese salt, 2.0~10.0mg of aluminium salt are heated molten
Solution, 120~130 DEG C of 10~20min of high pressure sterilization.
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| CN110846263A (en) * | 2019-12-31 | 2020-02-28 | 扬中酵诚生物技术研究有限公司 | High-density culture medium for escherichia coli |
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