CN1300302C - Culture medium for culturing eel vibrio - Google Patents
Culture medium for culturing eel vibrio Download PDFInfo
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- CN1300302C CN1300302C CNB021605882A CN02160588A CN1300302C CN 1300302 C CN1300302 C CN 1300302C CN B021605882 A CNB021605882 A CN B021605882A CN 02160588 A CN02160588 A CN 02160588A CN 1300302 C CN1300302 C CN 1300302C
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- Prior art keywords
- substratum
- vibrio anguillarum
- strain
- culture medium
- iron
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Abstract
The present invention provides a culture medium for culturing eel vibrio, which is especially suitable for culturing a defect strain of an iron ingesting system of the eel vibrio. The present invention comprises 0.08 to 0.12 mol/L of carbon source in C6H12O6 monomer, 15 to 75 mmol/L of inorganic nitrogen source in N, 5 to 100 mmol/L of inorganic phosphorus source in PO4<3->, 0.05 to 4 mmol/L of Mg<2+>, 0.5 to 10 g/L of organic nitrogen source, 18 to 30 g/L of sodium chloride and 0.1 to 1 mmol/L of iron ion, and pH value is 6.8 to 8.0.
Description
Technical field
The present invention relates to be used to cultivate the substratum of Vibrio anguillarum, especially be fit to the substratum of Vibrio anguillarum iron capturing system defective strain.
Background technology
Vibrio anguillarum is serious harm marine fishery production one a class important pathogen.Utilize the wild-type Vibrio anguillarum to make inactivated vaccine in the world and prevent that pathogenic bacterial infection from achieving success, the commercialization inactivated vaccine is used for many years.Compare the full complex medium of tradition, basal culture medium more helps the growth of wild-type Vibrio anguillarum, thereby helps the scale operation of Vibrio anguillarum inactivated vaccine.In addition, the Vibrio anguillarum iron capturing system defective strain that utilizes sophisticated in the world method mutagenesis screening to obtain has the value of potential attenuated live vaccine.Basal culture medium especially is fit to the growth of this defective strain, lays the foundation thereby promote for the commercialization of attenuated eel vibrio living vaccine.The strain of Vibrio anguillarum iron capturing system defective is the plasmid-free strain, and it is low to take the photograph the iron ability, compares with wild Vibrio anguillarum to have unique growth and metabolic characteristic.The special substratum of essential employing guarantees effective picked-up of ferric ion in the process of large scale culturing iron capturing system defective strain, and the equilibrium of various nutritive ingredients is keys of its cultivation.
The present relevant report of still the strain of Vibrio anguillarum iron capturing system defective not being cultivated, the cultivation of wild-type Vibrio anguillarum be mainly with an amount of NaCl of TGY culture medium supplemented, on the 30L reactor with batch training method large scale culturing.Under 28 ℃, through cultivating 24 hours, final bacterium output is 18g (thalline weight in wet base)/L (United States Patent (USP) U.S.P 3,862,313).
But with the culture medium culturing iron capturing system defective strain of above-mentioned report, have following deficiency: compositions such as the C in the substratum, N, P do not reach optimum concn and proportioning, and the defective strain can not reach best growth conditions, and substrate utilization efficient is low.Determine best ferric iron source, and to its concentration and exist environment to be optimized.Utilize peptone to cultivate, the cost height on commercial angle, is unfavorable for the large-scale promotion of defective strain as living vaccine.
Summary of the invention:
Technical problem
The present invention is intended to overcome the defective that the above known substratum is not suitable for the strain of Vibrio anguillarum iron capturing system defective, sets up a kind of Vibrio anguillarum wild-type large scale culturing of both can having improved, and can realize the efficient culture medium of Vibrio anguillarum defective strain large scale culturing again.
Technical scheme
In order to solve above technical problem, the invention provides a kind of substratum, wherein comprise: carbon source, press C
6H
12O
6The monomer meter, 0.08-0.12mol/L; Inorganic nitrogen-sourced, by N, 15-75mmol/L; Inorganic phosphorous sources is pressed PO
4 3-Meter, 5-100mmol/L; Mg
2+0.05-4mmol/L; Organic nitrogen source, 0.5-10g/L; Sodium-chlor, 18-30g/L; Fe
3+, 100-1000 μ mol/L; PH6.8-8.0.
Carbon source of the present invention can be selected from various known conventional carbon sources, for example sucrose, glucose, maltose, or its combination.Described inorganic nitrogen-sourced be selected from conventional inorganic nitrogen-sourced, for example ammonium chloride, ammonium sulfate, ammonium nitrate, or its combination.Described inorganic phosphorous sources is selected from conventional inorganic phosphorous sources, for example potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic, or its combination.Described organic nitrogen source is selected from yeast extract commonly used, peptone, hydrolysis casein food grade etc., or its combination.
In one of embodiment of the present invention, described substratum also contains micro-0-80 μ mol/L.Described trace element is selected from: bismuth (Bi
3+), zinc (Zn
2+), iron (Fe
3+), manganese (Mn
2+), calcium (Ca
2+), copper (Cu
2+), aluminium (Al
3+), nickel (Ni
2+), cobalt (Co
2+), or their combination.
In one of embodiment of the present invention, described substratum also comprises Tricine, 0-2g/L.
In one of embodiment of the present invention, described substratum comprises: sucrose 20g/L; Ammonium chloride 1.5g/L; Na
2HPO
412H
2O 9g/L, KH
2PO
40.8g/L; MgSO
46H
2O 0.2g/L; Yeast extract 3g/L; Sodium-chlor 25g/L; Ferric ammonium citrate 400 μ mol/L; Microelement concentrate 10ml/L; Tricine 0.7g/L; PH7.6; Described microelement concentrate comprises: H
3BO
30.2g/L, ZnSO
47H
2O 0.5g/L, MnSO
40.8g/L, CoCl
26H
2O 0.4g/L, CaCl
20.5g/L, CuSO
45H
2O 0.5g/L, FeSO
47H
2O 0.4g/L, AlCl
36H
2O 0.2g/L, NiCl
26H
2O 0.2g/L.
In one of embodiment of the present invention, what cultivated is the strain of Vibrio anguillarum iron capturing system defective.
Beneficial effect
Substratum of the present invention is with regard to C, N, P concentration and proportioning, and best ferric iron source and concentration thereof are optimized, and can improve the cultivation of Vibrio anguillarum.And, beat allly be that substratum of the present invention especially is fit to the high-density culture of iron capturing system defective strain, for developing laying the foundation of the full-synthetic culture medium that is fit to this defective strain.In addition, compare with the common complex medium of for example TGY, substratum of the present invention adopts raw materials such as simple inorganic salt, carbon source and yeast extract, main chemical compositions is clear, stay in grade, the stable and stdn that helps producing, and, the cost of unit output is lower, helps the large-scale promotion of Vibrio anguillarum iron capturing system defective strain as living vaccine.
Description of drawings
Fig. 1: the Vibrio anguillarum wild strain compares at the growing state of substratum of the present invention and known complex medium (TGY+0.25%NaCl in the United States Patent (USP) 3862313).
Fig. 2: the comparison that the strain of Vibrio anguillarum iron capturing system defective is gone up growing state at substratum of the present invention and common complex medium (TGY+0.25%NaCl in the United States Patent (USP) 3862313).
Embodiment
Below will further describe the present invention by embodiment.But it is pointed out that following explanation is only for for example, to the unqualified meaning of scope of the present invention.
Embodiment 1: culture medium preparation
According to table 1 formulated culture medium A, B, C and D.Wherein, D is the TGY+0.25%NaCl in the United States Patent (USP) 3862313.
Table 1:
| Culture medium A | Substratum B | Culture medium C | Substratum D | |||||
| Kind | Content | Kind | Content | Kind | Content | Kind | Content | |
| Carbon source | Sucrose | 20g/L | Glucose | 14g/L | Maltose | 22g/L | Glucose | 2.5g/L |
| Inorganic nitrogen-sourced | Ammonium chloride | 1.5g/L | Ammonium sulfate | 2g/L | Ammonium chloride | 4g/L | - | - |
| The phosphorus source | Na 2HPO 4·1 2H 2O | 9g/L | K 2HPO 4 | 0.75g/L | K 2HPO 4 | 15g/L | - | - |
| KH 2PO 4 | 0.8g/L | KH 2PO 4 | 0.09g/L | KH 2PO 4 | 1.8g/L | |||
| The magnesium source | MgSO 4· 7H 2O | 0.2g/L | MgCl 2· 6H 2O | 0.01g/L | MgSO 4· 7H 2O | 0.98g/L | - | - |
| Organic nitrogen source | Yeast extract | 3g/L | The hydrolysis casein food grade | 0.5g/L | Peptone | 10g/L | Yeast extract | 5g/L |
| Peptone | 10g/L | |||||||
| Sodium-chlor | 25g/L | 18g/L | 30g/L | 25g/L | ||||
| Ferric iron | Ferric ammonium citrate | 0.2g/L | FeCl 3·6H 2O | 0.027g/L | FeCl 3·6H 2O | 0.27g/L | - | - |
| Trace element | See Table 2 | See Table 2 | ||||||
| Tricine | 0.7g/L | - | - | 2.0g/L | - | - | ||
| pH | 7.5 | 6.8 | 8.0 | 7.5 | ||||
Table 2:
| Component | Content (g/L) | |
| Culture medium A | Culture medium C | |
| H 3BO 3 | 2 | 5 |
| ZnSO 4·7H 2O | 5 | 20 |
| MnSO 4·H 2O | 8 | 14 |
| CoCl 2·6H 2O | 4 | 19 |
| CaCl 2 | 5 | 8.9 |
| CuSO 4·5H 2O | 5 | 20 |
| FeSO 4·7H 2O | 4 | 22 |
| AlCl 3·6H 2O | 2 | 19 |
| NiCl 2·6H 2O | 2 | 19 |
Before all medium sterilizations, the pH value is adjusted to preset value.For prevent to precipitate and nutritional factor between destructive reaction, be divided into organic nutrient substance, inorganic salt, trace element and four parts of carbon source, autoclaving respectively, the special filtration sterilization of ferric salt solution.
Embodiment 2: the cultivation of Vibrio anguillarum wild strain
The Vibrio anguillarum wild strain that present embodiment is cultivated is Vibrio anguillarum (W-1, O1 serotype), be in the sick fish body of Huanghai Sea marine site, Shandong high-density breeding perch (Lateolabrax japonicus) outburst vibriosis, to separate to obtain, have strong vibriosis virulence.
According to component in the table 1 and content, preparation culture medium A and substratum D.Before the medium sterilization, the pH value is adjusted to 7.5.250ml shakes that the substratum liquid amount is 40ml in the bottle.Insert the fresh seeds (LB culture medium culturing) of cultivating through 12 hours then, inoculum density is 7.5%.Cultivated through 24 hours under 28 ℃, growth curve as shown in Figure 1.The growing state of Vibrio anguillarum wild strain in substratum of the present invention is better than the growth in the common complex medium (TGY+0.25%NaCl described in the USP3862313).
Embodiment 3: the preparation of Vibrio anguillarum iron capturing system defective strain
According to currently known methods, the Vibrio anguillarum wild strain among the embodiment 2 is carried out mutagenesis (Crosa, J.D., L.Hedges and M.Schiewe.1980.Infect.Immun.27:897-902 with the alternating temperature culture method; Tolmasky, M., L.Actis, A.Toranzo, J.Barja, and J.Crosa.1985.J.Gen.Microbiol.131:1987-1997.).In brief, get Vibrio anguillarum wild strain inclined-plane seed and be inoculated in the LB substratum, cultivate at 27 ℃ of following test tubes.The vigorous bacterium liquid of getting 12 hours of growth is inoculated in fresh LB substratum and (replenishes Fe
3+To 200 μ M), cultivated 12 hours for 37 ℃.With this bacterium liquid is bacterial classification, is inoculated in fresh benefit iron substratum, 37 ℃ of cultivations.As mentioned above, going down to posterity more than 5 generations at 37 ℃ is a mutagenesis cycle.
According to currently known methods, with the screening of CAS agar plate (Bernhard Schwyn, J.B.Neilands.1986..Analytical Biochemistry.160:47-56.) method.In brief, in generation, finished all after dates of above mutagenesis, gets gained bacterium liquid, is different multiples with the 3%NaCl solution dilution, is coated with the LB flat board respectively, treats that single bacterium colony grows.Each single bacterium colony is distinguished dibbling in sieve flat board and Fe
3+Abundant dull and stereotyped.On the sieve plate, can't grow and be potential plasmid disappearance strain (strain of iron capturing system defective), further alternating temperature mutagenesis what enrich dull and stereotyped normal growth.
With agarose electrophoresis and pcr amplification method above-mentioned potential plasmid being lacked strain identifies.In brief, with the plasmid DNA of alkaline lysis method of extracting mutagenic strain, extract scale 50ml bacterium liquid (OD
5500.4).Product is dissolved in the 50 μ l TE damping fluids, gets 10 μ l and do the electrophoresis checking.With the wild type strain nutrient solution of equal volume as positive control.To the negative strain of electrophoresis, do the PCR checking.Get that the fatDCBA gene is a target fragment on the pJM1 plasmid, the about 3kb of length, design primer.Reaction conditions: 94 ℃ of sex change 1 minute, 62.5 ℃ of renaturation 1.5 minutes, 72 ℃ were extended 3 minutes, 30 circulations.Getting 10 μ l, to do electrophoresis checking gained be the strain of Vibrio anguillarum iron capturing system defective.
Embodiment 4: the cultivation of Vibrio anguillarum iron capturing system defective strain
Described according to embodiment 1, the Vibrio anguillarum iron capturing system defective strain that embodiment 3 is made is incubated in A, B, C and the D substratum.Through 24 hours shake-flask culture, growth curve as shown in Figure 2 under 28 ℃.As shown in the figure, the growing state of Vibrio anguillarum iron capturing system defective strain in culture medium A-C of the present invention will obviously be better than the growth in the common complex medium (D), is best especially with the culture medium A.
Claims (5)
1. substratum that is used to cultivate Vibrio anguillarum wherein comprises: carbon source, press C
6H
12O
6The monomer meter, 0.08-0.12mol/L; Inorganic nitrogen-sourced, by N, 15-75mmol/L; Inorganic phosphorous sources is pressed PO
4 3-Meter, 5-100mmol/L; Mg
2+0.05-4mmol/L; Organic nitrogen source, 0.5-10g/L; Sodium-chlor, 18-30g/L; Fe
3+, 100-1000 μ mol/L; PH6.8-8.0.
2. substratum according to claim 1 also contains micro-0-80 μ mol/L, and described trace element is selected from: Bi
3+, Zn
2+, Fe
3+, Mn
2+, Ca
2+, Cu
2+, Al
3+, Ni
2+, Co
2+Or their combination.
3. substratum according to claim 1 and 2 also comprises Tricine, and its concentration is 0-2g/L.
4. substratum according to claim 3, wherein, described carbon source is sucrose 20g/L; Described inorganic nitrogen-sourced be ammonium chloride 1.5g/L; Described inorganic phosphorous sources is Na
2HPO
412H
2O 9g/L, KH
2PO
40.8g/L; Described Mg
2+Be MgSO
46H
2O 0.2g/L; Described organic nitrogen source is yeast extract 3g/L; Sodium-chlor 25g/L; Described Fe
3+Be ferric ammonium citrate 400 μ mol/L; Described trace element is microelement concentrate 10ml/L; Tricine 0.7g/L; PH7.6; Described microelement concentrate comprises: H
3BO
30.2g/L, ZnSO
47H
2O 0.5g/L, MnSO
40.8g/L, CoCl
26H
2O 0.4g/L, CaCl
20.5g/L, CuSO
45H
2O 0.5g/L, FeSO
47H
2O 0.4g/L, AlCl
36H
2O 0.2g/L, NiCl
26H
2O 0.2g/L.
5. the described substratum of claim 1 is used to cultivate the purposes of Vibrio anguillarum iron capturing system defective strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021605882A CN1300302C (en) | 2002-12-30 | 2002-12-30 | Culture medium for culturing eel vibrio |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021605882A CN1300302C (en) | 2002-12-30 | 2002-12-30 | Culture medium for culturing eel vibrio |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1511946A CN1511946A (en) | 2004-07-14 |
| CN1300302C true CN1300302C (en) | 2007-02-14 |
Family
ID=34237935
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021605882A Expired - Fee Related CN1300302C (en) | 2002-12-30 | 2002-12-30 | Culture medium for culturing eel vibrio |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1300302C (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3862313A (en) * | 1974-01-17 | 1975-01-21 | Us Interior | Vibrio vaccine and immunization |
| JPS5699423A (en) * | 1980-09-10 | 1981-08-10 | Kitasato Inst:The | Immune endowment to cultured fish by vaccine of vibrio disease |
-
2002
- 2002-12-30 CN CNB021605882A patent/CN1300302C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3862313A (en) * | 1974-01-17 | 1975-01-21 | Us Interior | Vibrio vaccine and immunization |
| JPS5699423A (en) * | 1980-09-10 | 1981-08-10 | Kitasato Inst:The | Immune endowment to cultured fish by vaccine of vibrio disease |
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| Publication number | Publication date |
|---|---|
| CN1511946A (en) | 2004-07-14 |
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Owner name: SHANGHAI HAOSI MARINE BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: EAST CHINA UNIVERSITY OF SCIENCE Effective date: 20100723 |
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Free format text: CORRECT: ADDRESS; FROM: 200237 NO.130, MEILONG ROAD, SHANGHAI CITY TO: 201306 NO.4, STREET A0201,JIANGANGHAIYANG HIGH-TECH INDUSTRIAL BASE, NANHUI DISTRICT, SHANGHAI CITY |
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Effective date of registration: 20100723 Address after: 201306, Nanhui District, Shanghai port hi tech industrialization base of the sea A0201 neighborhood No. 4 Patentee after: Shanghai HAOSI Marine Biotechnology Co. Ltd. Address before: 200237 No. 130, Meilong Road, Shanghai Patentee before: East China University of Science and Technology |
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Granted publication date: 20070214 Termination date: 20201230 |
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| CF01 | Termination of patent right due to non-payment of annual fee |