CN103695348B - Method for fermenting mycobacterium tuberculosis bacteria - Google Patents
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- CN103695348B CN103695348B CN201310692668.2A CN201310692668A CN103695348B CN 103695348 B CN103695348 B CN 103695348B CN 201310692668 A CN201310692668 A CN 201310692668A CN 103695348 B CN103695348 B CN 103695348B
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Abstract
The invention relates to a fermentation method of mycobacterium tuberculosis, belongs to the field of biological fermentation, and provides a bran-new method for fermenting mycobacterium tuberculosis. The results of ultra-high bacterial concentration of the mycobacterium tuberculosis and abundant expression of target protein are finally achieved by primary amplification, secondary amplification, primary fermentation and secondary fermentation of a bacterial strain. The bran-new fermentation method of the mycobacterium tuberculosis is obtained. The fermentation yield is not lower than 12g/L, the total rTPA (recombinant tissue plasminogen activator) 38 target protein in the bacteria can be up to over 15%, and the activity is high, which cannot be achieved by the prior art. The bacteria fermentation is large in batch quantity, and 10-200L of bacteria can be fermented one time. The method is fast in bacteria collection and high in production efficiency.
Description
Technical field
The present invention relates to the fermentation process of mycobacterium tuberculosis, belong to biological fermentation field.
Background technology
Tuberculosis infects by mycobacterium tuberculosis complex the multi organ infection disease caused, and is mainly pulmonary tuberculosis.Wherein patient newly sends out every year up to 1,500,000 in China.The EPDML key character of tubercule bacillus is the latent infection of mycobacterium tuberculosis.Mycobacterium tuberculosis latent infection is a kind of sub-clinical state, and without actinoscopy sign, bacterium is dormant state, about has the mycobacterium tuberculosis latent infection crowd of 10% can develop into tuberculosis patient in life.
Current tubercule bacillus latent infection crowd there is no diagnosis gold standard, the test of PPD skin test is the common method of diagnosing tubercle bacillus latent infection crowd, but this method has certain defective, because testing PPD antigen used is that mycobacterium tuberculosis, nonpathogenic mycobacteria and bacille Calmette-Guerin vaccine (i.e. BCG) are common, have cross reaction, specificity is poor.China is the country of general bcg vaccination, and be phenomenologically difficult to distinguish to the reaction that PPD tests due to BCG (Bacille Calmette-Guerin) vaccination person and tubercule bacillus natural infection person, even if adopt above-mentioned PPD skin test test strong positive as judging criterion, still can the infected of some BCG (Bacille Calmette-Guerin) vaccination person and nonpathogenic mycobacteria be included in tubercule bacillus latent infection crowd, the practical function therefore causing PPD to test in tubercule bacillus latent infection crowd diagnosis be very limited.The preparation that a kind of new specificity is high, side effect is little need be found replace.Restructuring Mycobacterium tuberculosis albumen 38K D a (r T P A38) is a kind of lipoprotein and the major antigen of Mycobacterium tuberculosis, and its effect of bringing out cavy delayed type hypersensitivity D T H is better than other single antigen.The Sensitivity and Specificity detected for tuberculosis serological is higher.Therefore, r T P A38 albumen is the albumen at present with better application prospect.
But, in the prior art, because mycobacterium tuberculosis is in the culturing process of cell, and expression target protein that can be a large amount of unlike intestinal bacteria, and there is the low defect of this folding activities with intestinal bacteria prediction scheme and expression r T P A38 albumen, therefore, the present inventor, based on the defect of prior art, devises the present invention.The present invention is by successive optimization mycobacterium tuberculosis cultural method of the prior art, provide a kind of brand-new mycobacterium tuberculosis fermentation process, increased by the one-level of bacterial strain, result that secondary amplification, one grade fermemtation, second order fermentation finally achieve the dense superelevation of mycobacterium tuberculosis bacterium, target protein great expression.
Summary of the invention
The object of the present invention is to provide: the method for mycobacterium tuberculosis thalline fermentation, thus collect a large amount of thalline increasing and obtain, obtain a large amount of target proteins, facilitate the use of r T P A38 albumen for extensive vaccine.
Technical scheme of the present invention: the method for mycobacterium tuberculosis thalline fermentation, comprises the steps: one, the preparation of one-level amplification liquid: get inclined-plane list bacterium colony, be placed in the test tube of one-level amplification culture medium, overnight incubation in full temperature shaking culture case; In Bechtop, add penbritin in every bottle of one-level amplification culture medium, then inoculate the bacterial classification in test tube, constant-temperature shaking culture in full temperature shaking culture case, it is about 1-2 that every bottle of sampling detects OD600 value;
Two, secondary amplification liquid: get primary seed solution and be inoculated in secondary amplification substratum, constant-temperature shaking culture in full temperature shaking culture case, it is 1-2 that every bottle of sampling detects OD600 value;
Three, one grade fermemtation is cultivated: before inoculation, setting ventilation is 2500L/h, and tank pressure is 0.04Mp, and stirring velocity is 560rpm, and dissolved oxygen when temperature is 37 DEG C is 100%.With the inoculum size of 5%-7% inoculation secondary amplification liquid in the 10L fermention medium in seeding tank, fermentation culture, cultivates 2-4h to OD
6002 ~ 3, the OD600 value of fermented liquid is measured in culturing process every half an hour;
Four, second order fermentation is cultivated: to be inoculated in fermentor tank in 120L fermention medium by satisfactory for OD600 in seeding tank one grade fermemtation bacterium liquid with the inoculum size of 5-10%; Continuous aerated culture received bacterium, every the OD600 value about measuring fermented liquid half an hour in culturing process in OD600 >=8 after about 4 to 5 hours;
Five, bacterium is received: adopt tubular-bowl centrifuge to carry out centrifugal bacterium liquid and abandon supernatant liquor, deposited in clean container by microorganism collection.
Six, quality index: smear gramstaining, observing under an optical microscope should be typical gram negative bacillus, in bacterial protein, (target protein rTPA38) is not less than 15%, fermentation yield: be not less than 12g/L, calculation formula: fermentation yield (g/L)=wet bacterium weight (g)/fermentation basic medium amount (L).
Beneficial effect of the present invention: the present inventor is by improving the fermentation process of mycobacterium tuberculosis, optimize the concrete conditional parameter of each step, obtain the fermentation process of brand-new mycobacterium tuberculosis, fermentation yield is not less than 12g/L, and total rTPA38 target protein can reach more than 15% in thalline, activity is also higher, and these are all that prior art is beyond one's reach.The batch of thalline fermentation is large, the disposable 10L to 200L that ferments, and receive bacterium speed fast, production efficiency is high.
Accompanying drawing explanation
Fig. 1 zymocyte liquid gramstaining opticmicroscope (typical gram negative bacillus)
Fig. 2 thalline fermentation process process flow sheet
Embodiment
Embodiment 1:
Substratum is prepared
(1) mycobacterium tuberculosis amplification culture medium is Roche egg slant medium, buys from Roche Holding Ag.
(2) fermention medium (1000mL): sodium selenate 10mg, potassium primary phosphate 2.4g, b) magnesium sulfate 0.24g, c) Citric Acid 0.6g, d) asparagine 3.6g, e) glycerine 20mL, f) yam starch 30g, g) fresh ovum gallinaceum liquid 940mL, h) Sodium Glutamate 0.15g surplus is water.
(3) preparation of other feed supplement liquid:
Dilute hydrochloric acid solution packing (1000mL): get each packing 500mL of dilute hydrochloric acid HCl1000mL and enter 2 500mL in sterilizing feed supplement bottle, liquor ammoniae fortis packing (1000mL): get each packing 500mL of liquor ammoniae fortis 1000mL and enter 2 500mL in sterilizing feed supplement bottle.
80% carbon source preparation (8L): taking that glycerine 6400g to be dissolved in purified water to cumulative volume is 8L, and be distributed in 5L feed supplement bottle, every bottle of 4L, 121 DEG C of sterilizing 20min, after cooling, normal temperature is airtight deposits.
50mg/mL penbritin (10mL): take 1g penbritin 20mL purified water and dissolve.In Bechtop, degerming with aseptic syringe needle metre filter, be distributed in sterilising vessel, packing specification is 1mL/ pipe (1.5ml EP manage), totally 10 manages, preserves stand-by in 2-8 DEG C of refrigerator.
Embodiment 2: thalline fermentation process
One, one-level amplification liquid preparation: get the mycobacterium tuberculosis (bacterial strain that this area is conventional, such as: ATCC93009) inclined-plane list bacterium colony, be placed in the test tube containing 3mL amplification culture medium, in full temperature shaking culture case, constant temperature 37 ± 1 DEG C, rotating speed 170r/min shaking culture are spent the night; In every bottle of amplification culture medium (8mL), 50mg/mL penbritin 8 μ l is added in Bechtop, then the bacterial classification in 80 μ l test tubes is inoculated, in full temperature shaking culture case after constant temperature 37 ± 1 DEG C, rotating speed 170r/min shaking culture 10-12h, every bottle of sampling 1-2mL, purified water detects with visible spectrophotometer and calculates OD600 value after suitably diluting and is about 1;
Two, secondary amplification liquid: before inoculation, in 2 bottles of amplification culture mediums (500mL), 50mg/mL penbritin 500 μ l is added in Bechtop, then getting primary seed solution 5mL is inoculated in secondary amplification substratum, in full temperature shaking culture case after constant temperature 37 ± 1 DEG C, rotating speed 170r/min shaking culture 12-14h, every bottle of sampling 1-2mL, purified water detects with visible spectrophotometer and calculates OD600 value after suitably diluting and is about 1.5;
Three, one grade fermemtation is cultivated: (1) electrode alignment: pH electrode corrects, and before seed culture medium sterilizing, at room temperature corrects with the reference liquid of pH6.86 and pH9.18.The setting of DO electrode: before substratum preparation, DO electrode being put into saturated sodium bisulfite solution, to set DO value be 0; Before inoculation, setting ventilation is 2500L/h, and tank pressure is 0.04Mp, and stirring velocity is 560rpm, and dissolved oxygen when temperature is 37 DEG C is 100%.(2) work before inoculation: connect 500mL liquor ammoniae fortis (concentration about 28%) solution feed supplement bottle and 500mL dilute hydrochloric acid (concentration about 10%) solution feed supplement bottle, regulates the pH to 6.8-7.0 of substratum; (3) inoculate: satisfactory for OD600 secondary amplification liquid is inoculated secondary amplification liquid in the 10L fermention medium in seeding tank with the inoculum size of 5%-7% under flame protection; (4) fermentation culture: culture temperature 37 ± 1 DEG C, initial stirring velocity is 100rpm, and air flow is 300L/h, tank pressure 0.02-0.04Mpa, pH6.8-7.0, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid adjustment pH, and dissolved oxygen setting 35%, controls between 30%-40%, dissolved oxygen and rotating speed coupling, if dissolved oxygen is lower than 30%, strengthen air flow, cultivate 2-4h to OD
6002 ~ 3, the OD600 value of fermented liquid is measured in culturing process every half an hour;
Four, second order fermentation is cultivated: (1) electrode alignment: pH electrode corrects: before medium sterilization, corrects pH electrode with the reference liquid of pH6.86 and pH9.18.The setting of DO electrode: before substratum preparation, DO electrode being put into saturated sodium bisulfite solution, to set DO value be 0; Before inoculation, setting ventilation is 16m
3/ h, tank pressure is 0.04Mp, and stirring velocity is 400rpm, and dissolved oxygen when temperature is 37 ± 1 DEG C is 100%; (2) work before inoculation: connect 500mL liquor ammoniae fortis feed supplement bottle, 500mL rare HCl solution feed supplement bottle, 4L80% carbon source feed supplement bottle; (3) inoculate: satisfactory for OD600 in seeding tank one grade fermemtation liquid to be inoculated in fermentor tank in 120L fermention medium with the inoculum size of 5-10%; (4) fermentation culture: initial stirring velocity is 100rpm, and air flow is 1.8m
3/ h, pH6.8-7.0,37 ± 1 DEG C of cultivations, after dissolved oxygen drops to 35%, setting DO 35% associate with rotating speed and controls, and control at 30%-40%, if association control dissolved oxygen is still lower than 30%, cancellation associates, adjustable rotating speed and tune up air flow gradually, is maximumly adjusted to 16m
3/ h, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid solution to regulate pH, dissolved oxygen set(ting)value is 15%, when OD600>=3,, adjusting rotary speed controls dissolved oxygen at 10%-20%, and air flow still keeps the air flow before inducing, when DO significantly rises, add 80% carbon source of sterilizing with carbon source unrestricted model stream.37 ± 1 DEG C are continued continuous aerated culture and receive bacterium, every the OD600 value about measuring fermented liquid half an hour in culturing process in OD600 >=8 after about 4 to 5 hours;
Five, bacterium is received: adopt tubular-bowl centrifuge to carry out centrifugal bacterium liquid with 45Hz frequency (about 14000rpm) and abandon supernatant liquor, flow velocity is 1.4-1.8L/min, is deposited in clean container by microorganism collection.
Six, quality index: smear gramstaining, the thalline observed under an optical microscope after fermentation is typical gram negative bacillus, detect that in bacterial protein, rTPA38 albumen is 19% by SDS-PAGE, fermentation yield: be 12g/L, calculation formula: fermentation yield (g/L)=wet bacterium weight (g)/fermentation basic medium amount (L).
The cultural method cultivation mycobacterium tuberculosis that embodiment 3 is common
Get inclined-plane list bacterium colony, be placed in the test tube containing 3mL Roche egg slant medium, in full temperature shaking culture case, constant temperature 37 ± 1 DEG C, rotating speed 170r/min shaking culture are spent the night; In every bottle of amplification culture medium (8mL), 50mg/mL penbritin 8 μ l is added in Bechtop, then the bacterial classification in 80 μ l test tubes is inoculated, in full temperature shaking culture case after constant temperature 37 ± 1 DEG C, rotating speed 170r/min shaking culture 10-12h, every bottle of sampling 1-2mL, purified water detects with visible spectrophotometer and calculates OD600 value after suitably diluting and is about 1; Be inoculated in the 10L Roche egg slant medium substratum in seeding tank with the inoculum size of 5%-7%, adopt conventional cultural method, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid adjustment pH, cultivate through about 12h, fermentation ends, fermentation yield: be 3g/L, in bacterial protein, target protein rTPA38 is 15%.
Claims (1)
1. the method for mycobacterium tuberculosis thalline fermentation, comprises the following steps:
One, one-level amplification liquid preparation: get mycobacterium tuberculosis inclined-plane list bacterium colony, be placed in the test tube containing 3mL amplification culture medium, constant temperature 37 ± 1 DEG C, rotating speed 170r/min shaking culture are spent the night in full temperature shaking culture case; In the triangular flask containing 8mL amplification culture medium, 50mg/mL penbritin 8 μ l is added in Bechtop, then the bacterial classification in 80 μ l test tubes is inoculated, in full temperature shaking culture case after constant temperature 37 ± 1 DEG C, rotating speed 170r/min shaking culture 10-12h, sampling, to OD
600value is about 1 and can stops cultivating;
Two, secondary amplification liquid: before inoculation, 50mg/mL penbritin 500 μ l is added to 2 bottles containing in the triangular flask of 500mL amplification culture medium in Bechtop, then getting one-level amplification liquid 5mL is inoculated in secondary amplification substratum, in full temperature shaking culture case after constant temperature 37 ± 1 DEG C, rotating speed 170r/min shaking culture 12-14h, sampling, to OD
600value is about 1.5;
Three, one grade fermemtation is cultivated: before (1) inoculation, setting ventilation is 2500L/h, and tank pressure is 0.04Mp, and stirring velocity is 560rpm, and dissolved oxygen when temperature is 37 DEG C is 100%; (2) work before inoculation: connecting 500mL concentration is 28% liquor ammoniae fortis feed supplement bottle and 500mL concentration 10% dilute hydrochloric acid solution feed supplement bottle, regulates the pH to 6.8-7.0 of substratum; (3) inoculate: be inoculated in the 10L fermention medium in seeding tank with the inoculum size of 5%-7%; (4) fermentation culture: culture temperature 37 ± 1 DEG C, initial stirring velocity is 100rpm, and air flow is 300L/h, tank pressure 0.02-0.04Mpa, pH6.8-7.0, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid adjustment pH, and dissolved oxygen setting 35%, controls between 30%-40%, dissolved oxygen and rotating speed coupling, if dissolved oxygen is lower than 30%, strengthen air flow, cultivate 2-4h to OD
6002 ~ 3, the OD of fermented liquid is measured in culturing process every half an hour
600value;
Four, second order fermentation is cultivated: before (1) inoculation, setting ventilation is 16m
3/ h, tank pressure is 0.04Mp, and stirring velocity is 400rpm, and dissolved oxygen when temperature is 37 ± 1 DEG C is 100%; (2) work before inoculation: connecting 500mL concentration is 28% liquor ammoniae fortis feed supplement bottle, 500mL concentration 10% rare HCl solution feed supplement bottle, 4L80% carbon source feed supplement bottle; (3) inoculate: by the OD after one grade fermemtation
600satisfactory seed liquor to be inoculated in fermentor tank in 120L fermention medium with the inoculum size of 5-10%; (4) fermentation culture: initial stirring velocity is 100rpm, and air flow is 1.8m
3/ h, pH6.8-7.0,37 ± 1 DEG C of cultivations, after dissolved oxygen drops to 35%, setting DO 35% associate with rotating speed and controls, and control at 30%-40%, if association control dissolved oxygen is lower than 30%, cancellation associates, and turns speed and tunes up air flow gradually, is maximumly adjusted to 16m
3/ h, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid solution to regulate pH; Work as OD
600when>=3, dissolved oxygen set(ting)value is 15%, and adjusting rotary speed controls dissolved oxygen at 10%-20%, and air flow is constant, when DO significantly rises, adds 80% carbon source of sterilizing with carbon source unrestricted model stream, and 37 ± 1 DEG C are continued continuous aerated culture OD after 4 to 5 hours
600>=8 receive bacterium, every the OD about measuring fermented liquid half an hour in culturing process
600value;
Receive bacterium: adopt tubular-bowl centrifuge to carry out centrifugal bacterium liquid with 45Hz frequency, 14000rpm and abandon supernatant liquor, flow velocity is 1.4-1.8L/min, is deposited in by microorganism collection in clean container, namely obtains the object thalline fermented;
Five, substratum is formulated as follows:
(1) mycobacterium tuberculosis amplification culture medium is Roche egg slant medium, buys from Roche Holding Ag;
(2) fermention medium 1000mL: sodium selenate 10mg, potassium primary phosphate 2.4g, magnesium sulfate 0.24g, Citric Acid 0.6g, asparagine 3.6g, glycerine 20mL, yam starch 40g, fresh ovum gallinaceum liquid 930mL, Sodium Glutamate 0.15g, surplus is water;
(3) preparation of other feed supplement liquid:
Dilute hydrochloric acid solution packing 1000mL: get each packing 500mL of dilute hydrochloric acid HCl 1000mL and enter 2 500mL in sterilizing feed supplement bottle, liquor ammoniae fortis packing 1000mL: get each packing 500mL of liquor ammoniae fortis 1000mL and enter 2 500mL in sterilizing feed supplement bottle;
80% carbon source preparation: taking that glycerine 6400g to be dissolved in purified water to cumulative volume is 8L, and be distributed in 5L feed supplement bottle, every bottle of 4L, 121 DEG C of sterilizing 20min, after cooling, normal temperature is airtight deposits;
50mg/mL penbritin 10mL: take 1g penbritin 20mL purified water and dissolve; In Bechtop, degerming with aseptic syringe needle metre filter, be distributed in sterilising vessel, packing specification is that 1mL/1.5ml EP manages, and totally 10 pipes, preserve stand-by in 2-8 DEG C of refrigerator.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423807A (en) * | 2007-11-02 | 2009-05-06 | 中国科学院沈阳应用生态研究所 | Method for preparing polycyclic aromatic hydrocarbon degrading bacteria |
| CN102295687A (en) * | 2011-08-12 | 2011-12-28 | 广东瀚森生物药业有限公司 | Renaturing and purifying method of mycobacterium-tuberculosis recombinant inclusion-body protein |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423807A (en) * | 2007-11-02 | 2009-05-06 | 中国科学院沈阳应用生态研究所 | Method for preparing polycyclic aromatic hydrocarbon degrading bacteria |
| CN102295687A (en) * | 2011-08-12 | 2011-12-28 | 广东瀚森生物药业有限公司 | Renaturing and purifying method of mycobacterium-tuberculosis recombinant inclusion-body protein |
Non-Patent Citations (3)
| Title |
|---|
| Liquid culture for Mycobacterium tuberculosis: proceed, but with caution;Anthony RM et.al.;《Int J Tuberc Lung Dis.》;20090930;第13卷(第9期);1051-3 * |
| 对罗氏培养基进一步改良的初步研究;于滨;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20090215;全文 * |
| 结核分枝杆菌早期培养滤液蛋白的制备和应用;都伟欣 等;《中国医药生物技术》;20120630;第7卷(第3期);229-231 * |
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