CN105296407A - Method for culturing avibacterium paragallinarum bacterial solution - Google Patents
Method for culturing avibacterium paragallinarum bacterial solution Download PDFInfo
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- CN105296407A CN105296407A CN201510896740.2A CN201510896740A CN105296407A CN 105296407 A CN105296407 A CN 105296407A CN 201510896740 A CN201510896740 A CN 201510896740A CN 105296407 A CN105296407 A CN 105296407A
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- Prior art keywords
- seed solution
- chicken fowl
- strain
- bacterium liquid
- enlarged culturing
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- 230000001580 bacterial effect Effects 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000012258 culturing Methods 0.000 title claims abstract description 18
- 241000606767 Avibacterium paragallinarum Species 0.000 title abstract 4
- 239000007788 liquid Substances 0.000 claims abstract description 21
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- 241000287828 Gallus gallus Species 0.000 claims description 29
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 18
- 241001037822 Bacillus bacterium Species 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 abstract description 12
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 229960005486 vaccine Drugs 0.000 abstract description 2
- 230000003321 amplification Effects 0.000 abstract 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 2
- 238000009776 industrial production Methods 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 8
- 239000002054 inoculum Substances 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000021049 nutrient content Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 235000000053 special nutrition Nutrition 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for culturing an avibacterium paragallinarum bacterial solution, and belongs to the field of fermentation culture. The method comprises the following steps: (1) preparing a primary seed solution; (2) preparing a secondary seed solution; (3) performing amplification culture on a microbial fermentation tank, wherein the secondary seed solution is an avibacterium paragallinarum WF strain or YC strain secondary seed solution, and the number of living bacteria is more than or equal to 4.5*10<9>CFU/ml after amplification culture. The method has the advantages of large number of living bacteria and short culture cycle, and can be used for providing an excellent process foundation to large-scale production of qualified bacterial liquid of avibacterium paragallinarum and greatly increasing and improving the industrial production process of vaccines.
Description
Technical field
The present invention relates to fermentation culture field, more specifically to a kind of cultural method of secondary chicken fowl bacillus bacterium liquid.
Background technology
Substratum is to provide the mixture of multiple nutrients material required for microbial growth and the various meta-bolites of synthesis, that prepare by a certain percentage, for obtaining the biological products of high-quality, high density, must nutritiously enrich complete, composition rationally, steady quality, the ability of producing bacterial classification anabolite can be given full play to.
The life characteristics of secondary chicken fowl bacillus is more fragile, it is the incomplete gram negative bacillus of class of enzymes system, growth not only needs the Carbon and nitrogen sources enriched, also need the special nutrition materials such as healthy animal serum, yeast leach liquor and coenzyme, and culture condition is harsh, bring difficulty to the fermentation culture of bacterium liquid, the viable count that enlarged culturing obtains of current secondary chicken fowl bacillus is not high, and culture cycle is long.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of cultural method of secondary chicken fowl bacillus bacterium liquid.
For achieving the above object, the present invention is by the following technical solutions:
A cultural method for secondary chicken fowl bacillus bacterium liquid, comprises the following steps: the preparation of (1) primary seed solution; (2) preparation of secondary seed solution; (3) microorganism fermentation tank enlarged culturing; It is characterized in that, described secondary seed solution is the WF strain of secondary chicken fowl bacillus or YC strain secondary seed solution, and after described enlarged culturing, each bacterial strain viable count all>=4.5 × 10
9cFU/mL.
Further, in described enlarged culturing, secondary chicken fowl bacillus secondary seed solution access amount is 5% (V/V).
Further, described enlarged culturing used medium is semisynthetic medium.
Further, the pH value of described semisynthetic medium is 7.2 ~ 7.6.
Further, described enlarged culturing is aerated culture, and air flow is 5 ~ 6L/ minute.
Further, described enlarged culturing condition is stirring velocity 100 ~ 200 revs/min, and culture temperature is 37 DEG C, and incubation time is 16-18h.
Beneficial effect of the present invention is: it is high that this culture process obtains bacterial strain viable count, and with short production cycle, improves a lot and improve the industrialized manufacturing technique of vaccine.
Embodiment
The following detailed description of specific embodiment of the invention; what be necessary to herein means out is; below implement just to further illustrate for of the present invention; limiting the scope of the invention can not be interpreted as; some nonessential improvement and adjustment that this art skilled person makes the present invention according to the invention described above content, still belong to protection scope of the present invention.
Bacterial classification: the WF strain of secondary chicken fowl bacillus and YC strain, Hua Hong biotechnology company limited provides by Shandong.
Substratum: semisynthetic medium, is made by oneself by Shandong Hua Hong biotechnology company limited laboratory.
Fermentor tank: 40L microorganism fermentation tank, purchased from Shanghai Gaoji Bioengineering Co., Ltd..
The preparation of embodiment 1 secondary seed solution
By the first order seed streak inoculation of the WF strain of secondary chicken fowl bacillus or YC strain in chicken broth agar plates, containing 5% ~ 10%CO
2condition under, 37 DEG C cultivate 16 ~ 18h, the colonies typical selecting fluorescence strong is inoculated in chicken soup culture medium, put 37 DEG C cultivate 16 ~ 20h, after pure inspection is qualified, be secondary seed, 2 ~ 8 DEG C of preservations, must not more than 6 ~ 8h.
Embodiment 2 secondary seed inoculum size is on the impact of seedling bacterial concentration
In semisynthetic medium, the secondary seed solution of the WF strain of secondary chicken fowl bacillus or YC strain is inoculated respectively by the inoculum size (V/V) of 1%, 2%, 3%, 5%, cultivate 20h for 37 DEG C, respectively live bacterial count is carried out to the bacterium liquid of different time sections, compare secondary seed different vaccination amount to the impact of seedling bacterial concentration.
Different secondary seed inoculum size cultivates concentration to the WF strain of secondary chicken fowl bacillus or YC strain bacterium liquid certain influence, and detailed results is in table 1 and table 2.
The different secondary seed inoculum size of table 1 is on the impact of WF strain bacterial concentration
The different secondary seed inoculum size of table 2 is on the impact of YC strain bacterial concentration
From table 1 and table 2, inoculate the secondary seed of 5% content in the medium, can obtain good culture effect within a short period of time, production cycle and production technique are considered, select 5% inoculum size for suitable inoculum size, incubation time is 16-18h.
The different training method of embodiment 3 is on the impact of seedling bacterial concentration
Respectively with quiescent culture and aerated culture mode, measure different bacterium liquid training method to the impact of bacterial concentration.Ventilation is in a small amount started during aerated culture, increase air flow gradually, air flow controls at 5 ~ 6L/ minute, cultivates 18h under 37 DEG C of conditions, carry out live bacterial count to the point in time sampling of 12h, 16h and 18h respectively, more different bacterium liquid training method is on the impact of bacterial concentration.
Quiescent culture and aerated culture are cultivated bacterium liquid live bacterial count to WF strain or YC strain and be there is considerable influence, and detailed results is in table 3.
The cultivation bacterium liquid live bacterial count result (× 10 of table 3 quiescent culture and aerated culture
9cFU/mL)
Secondary chicken fowl bacillus is in cultivation breeding, the meta-bolites enrichment produced, suppress the growing multiplication of thalline, aerated culture constantly inputs fresh air in culturing process, adjust the nutritive ingredient of substratum, uniformity coefficient and pH value, the advantage of therefore carrying out aerated culture to secondary chicken fowl bacillus is obvious.
The optimization of embodiment 4 microorganism fermentation tank enlarged culturing condition
Semisynthetic medium is added by the minimum loading amount of fermentor tank, the secondary seed solution of the WF strain of secondary chicken fowl bacillus or YC strain is added by 5% (V/V) of substratum total amount, carry out the independent enlarged culturing of microorganism fermentation tank, temperature controls at 37 DEG C, regulate mixing speed, pH value, air flow, cultivate 18h, carry out live bacterial count, the bacterial concentration of secondary chicken fowl bacillus under more different culture condition.
1. different stirring velocity test-results that bacterial concentration is affected
Cultivate in the process of the WF strain of secondary chicken fowl bacillus or YC strain utilizing microorganism fermentation tank, stirring velocity significantly can increase the concentration of bacterium liquid, but secondary chicken fowl bacillus is more fragile, too high stirring velocity can affect its growing multiplication, therefore comprehensive various factors, when microorganism fermentation tank is cultivated by we, it is 100 ~ 200 revs/min that stirring velocity controls, and detailed results is in table 4.
The different stirring velocity of table 4 microorganism fermentation tank is on the impact (× 10 of bacterium liquid final concentration
9cFU/mL)
2. different air flow test-results that bacterial concentration is affected
In the process that microorganism fermentation tank cultivates the WF strain of secondary chicken fowl bacillus or YC strain, continuous input fresh air, and get rid of bacteriogenic gas, the uniformity coefficient of timely adjustment medium nutrient content and pH value, larger on bacterial concentration impact, by test-results, we think that the air flow of 5 ~ 6L/ minute is better, and detailed results is in table 5.
The different air flow of table 5 is on the impact of bacterium liquid final concentration
Claims (6)
1. a cultural method for secondary chicken fowl bacillus bacterium liquid, comprises the following steps: the preparation of (1) primary seed solution; (2) preparation of secondary seed solution; (3) microorganism fermentation tank enlarged culturing; It is characterized in that, described secondary seed solution is the WF strain of secondary chicken fowl bacillus or YC strain secondary seed solution, and after described enlarged culturing, each bacterial strain viable count all>=4.5 × 10
9cFU/mL.
2. the cultural method of a kind of secondary chicken fowl bacillus bacterium liquid according to claim 1, it is characterized in that, in described enlarged culturing, secondary chicken fowl bacillus secondary seed solution access amount is 5% (V/V).
3. the cultural method of a kind of secondary chicken fowl bacillus bacterium liquid according to claim 1, it is characterized in that, described enlarged culturing used medium is semisynthetic medium.
4. the cultural method of a kind of secondary chicken fowl bacillus bacterium liquid according to claim 3, it is characterized in that, the pH value of described semisynthetic medium is 7.2 ~ 7.6.
5. the cultural method of a kind of secondary chicken fowl bacillus bacterium liquid according to claim 1, it is characterized in that, described enlarged culturing is aerated culture, and air flow is 5 ~ 6L/ minute.
6. the cultural method of a kind of secondary chicken fowl bacillus bacterium liquid according to claim 5, it is characterized in that, described enlarged culturing condition is stirring velocity 100 ~ 200 revs/min, and culture temperature is 37 DEG C, and incubation time is 16-18h.
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| CN201510896740.2A CN105296407A (en) | 2015-12-07 | 2015-12-07 | Method for culturing avibacterium paragallinarum bacterial solution |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925517A (en) * | 2016-07-21 | 2016-09-07 | 山东滨州沃华生物工程有限公司 | Serum-free anaerobic high-density fermentation culture process for Streptococcus equi subsp. zooepidemicu |
| CN110747148A (en) * | 2019-12-02 | 2020-02-04 | 天津瑞普生物技术股份有限公司 | Preparation method of avicenobacter paragallinarum culture medium |
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| CN103122324A (en) * | 2012-06-18 | 2013-05-29 | 山东华宏生物工程有限公司 | Method for culturing chicken escherichia coli |
| CN103667115A (en) * | 2013-11-18 | 2014-03-26 | 魏锁成 | Cultural method of haemophilus paragallinarum |
| CN104328077A (en) * | 2014-11-18 | 2015-02-04 | 北京华都诗华生物制品有限公司 | Avibacterium paragallinarum fermentation culture method |
-
2015
- 2015-12-07 CN CN201510896740.2A patent/CN105296407A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122324A (en) * | 2012-06-18 | 2013-05-29 | 山东华宏生物工程有限公司 | Method for culturing chicken escherichia coli |
| CN103667115A (en) * | 2013-11-18 | 2014-03-26 | 魏锁成 | Cultural method of haemophilus paragallinarum |
| CN104328077A (en) * | 2014-11-18 | 2015-02-04 | 北京华都诗华生物制品有限公司 | Avibacterium paragallinarum fermentation culture method |
Non-Patent Citations (2)
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| 周燕芬等: "副鸡嗜血杆菌（Hpg-8株）培养条件的优化及免疫原性的测定", 《广东畜牧兽医科技》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925517A (en) * | 2016-07-21 | 2016-09-07 | 山东滨州沃华生物工程有限公司 | Serum-free anaerobic high-density fermentation culture process for Streptococcus equi subsp. zooepidemicu |
| CN105925517B (en) * | 2016-07-21 | 2019-10-18 | 山东滨州沃华生物工程有限公司 | Malian drainage serum-free anaerobism high density fermentation culture technique |
| CN110747148A (en) * | 2019-12-02 | 2020-02-04 | 天津瑞普生物技术股份有限公司 | Preparation method of avicenobacter paragallinarum culture medium |
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Application publication date: 20160203 |