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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- seed solution
- chicken fowl
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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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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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| 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 |
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Patent Citations (3)
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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 |
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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 |