CN105039227A - A-group Neisseria meningitidis cultivation method - Google Patents

A-group Neisseria meningitidis cultivation method Download PDF

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Publication number
CN105039227A
CN105039227A CN201510543425.1A CN201510543425A CN105039227A CN 105039227 A CN105039227 A CN 105039227A CN 201510543425 A CN201510543425 A CN 201510543425A CN 105039227 A CN105039227 A CN 105039227A
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bacterial classification
seed
medium
group
purifying
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朱冲
米强
陈道远
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Chengdu Olymvax Biopharmaceuticals Inc
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Chengdu Olymvax Biopharmaceuticals Inc
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Priority to CN201510543425.1A priority Critical patent/CN105039227A/en
Publication of CN105039227A publication Critical patent/CN105039227A/en
Priority to PCT/CN2016/078086 priority patent/WO2017036140A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/36—Neisseria

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  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses an A-group Neisseria meningitidis cultivation method. The problem that the efficiency of directly extracting polysaccharide from A-group Neisseria meningitidis in the prior art is low and the extracted polysaccharide is not easily purified is solved. The A-group Neisseria meningitidis cultivation method comprises the following steps that (1) purified strains of a batch of working seeds are prepared; (2) the strains of the batch of working seeds are opened and inoculated into a semi-synthetic culture medium, cultivation is performed at the temperature of 35-37 DEG C for 16 to 24 hours, and then an appropriate quantity of seeds for production are prepared through three-generation multiplication culture; (3) the seeds for production are inoculated into a fermentation tank and are cultivated for 6 to 12 hours. The culture medium in the fermentation tank is the semi-synthetic culture medium, the temperature ranges from 35 DEG C to 37 DEG C, the pH value of the culture medium is 7.0 to 7.2, the stirring speed in the fermentation tank is 140 r/min to 150 r/min, and the inoculation quantity is 100,000 ml to 150,000 ml. The A-group Neisseria meningitidis cultivation method has the advantages that dead bacteria are little, polysaccharide is more easily purified, the polysaccharide extraction rate is higher and the like.

Description

The cultural method of A group meningitis cocci
Technical field
The present invention relates to a kind of cultural method of bacterial classification, what be specifically related to is the cultural method of A group meningitis cocci.
Background technology
Meningococcal meningitis (being called for short epidemic meningitis, lower same) is a kind of Acute respiratory infectious disease caused by the scorching coccus (Neisseriameningitidis) of neisseria meningitis.Over more than 100 year, always popular all over the world or be dispersed in generation, septicemia, meningitis can be caused after infection pathogen.Susceptible population is mainly children, the highest with fulminant type case fatality rate, can reach 40% ~ 60%.Each continent, world today sickness rate is 1,/10 ten thousand ~ 10,/10 ten thousand, and total case fatality rate, 5% ~ 15%, has neural system sequela up to the meningitis patient of 20%, comprises intellectual impairment and deafness etc.Carry out serological classification according to capsular polysaccharide type and can divide 13 serotypes, wherein A group, B group, C group account for 90% of Epidemic bacterial flora.A group meningitis cocci is more pandemic principal causative serogroups, particularly at so-called Africa " the popular band of epidemic meningitis ", just there will be once comparatively be very popular every 7-14.
China in 1938,1949, once to there are 5 national epidemic meningitis in nineteen fifty-nine, 1967 and 1977 popular; Wherein popular serious with spring in 1967, sickness rate is up to 4,03/,100,000, and case fatality rate is 5.49%.The case in China's past more than 90% is that A group germ is caused a disease, and present B group or C group germ also cause epidemic meningitis to break out sometimes.2003 start, and the sickness rate of C group's epidemic meningitis obviously rises.Current A group and C group account for more than 50% of all serogroupss altogether, and C group still has the trend increased further.Therefore, the emphasis of current China prevention epidemic meningitis work prevents A group and C group to be master.
The most effective means of current prevention meningococcal meningitis is vaccination.Research shows, the capsular polysaccharide of A group C group meningitis cocci has good immunogenicity, extracts capsular polysaccharide and directly can be prepared into vaccine, and the crowd after injecting immune can adaptive immune protection.China included vaccine of epidemic menigitis in National immunization Program from 2007, in the whole country to the universal inoculation of school-age children.The epidemic meningitis polysaccharide vaccine including National Immunization Program at present in comprises A meningococcal polysaccharide vaccine, A group C meningococcal polysaccharide vaccine.
Be all from A group meningitis cocci, extract polysaccharide make polysaccharide vaccine in prior art, and then reach the meningitic effect of prevention.Because polysaccharide extracts from pod membrane, if dead bacterium is more in spawn culture process, easily cause the extraction efficiency directly extracting polysaccharide from A group meningitis cocci low, and the polysaccharide extracted is not easy purifying.
Summary of the invention
The object of the invention is to solve the extraction efficiency directly extracting polysaccharide in prior art from A group meningitis cocci low, and the polysaccharide extracted is not easy the problem of purifying, provides a kind of cultural method of the A group meningitis cocci solved the problem.
For solving above-mentioned shortcoming, technical scheme of the present invention is as follows:
The cultural method of A group meningitis cocci, comprises the following steps:
(1) the working seed lots bacterial classification of purifying is prepared;
(2) open working seed lots bacterial classification, be seeded in half synthetic medium, cultivate 16 ~ 24 hours under temperature 35 ~ 37 DEG C of conditions, then through three generations's amplification cultivation, the production seed that preparation quantity is suitable;
(3) production seed is seeded to fermentor cultivation 6 ~ 12 hours; In fermentor tank, substratum is half synthetic medium, and temperature is 35 ~ 37 DEG C, and medium pH is 7.0 ~ 7.2, and in fermentor tank, stirring velocity is 140 ~ 150r/min, and inoculum size is 10 ~ 150,000/ml.
Preferably, the detailed process of described step (1) is as follows:
By A group meningitis Neisseria gonorrhoeae strain inoculation on pure medium, 20h is cultivated under 38 DEG C of conditions, select stalwartness and inoculate on pure medium without the bacterial classification of living contaminants and carry out second incubation, 12h is cultivated again under 38 DEG C of conditions, select stalwartness and bacterial classification without living contaminants adds in germfree defatted milk, mixing freeze-drying is prepared and obtains the working seed lots bacterial classification of purifying.
As preferred embodiment, the opening process of described purifying seed is as follows:
Working seed lots bacterial classification is dissolved in sterilized water, is seeded on 10% SBA substratum, be put under the environment of 35 ~ 37 DEG C and cultivate after 16 ~ 20 hours, pick out stalwartness and without the bacterial classification of living contaminants.
Further, the concrete cultivating process of described step (2) is as follows:
Purifying seed after unlatching is inoculated in half comprehensive liquid nutrient medium and carries out activation culture; The condition of activation culture is temperature 35 ~ 37 DEG C, pH7.0 ~ 7.2, stirring velocity 140 ~ 150r/min, and incubation time is 6 ~ 8h; Finally by the purifying seed after activation through three generations's amplification cultivation, the production seed that quantity is suitable can be prepared.
Further, the condition of described three generations's amplification cultivation is: amplification cultivation substratum is in half comprehensive liquid nutrient medium; Breeding condition is temperature 35 ~ 37 DEG C, pH7.0 ~ 7.2, stirring velocity 140 ~ 150r/min.
In order to reach best effect, the composition and ratio of described half comprehensive liquid nutrient medium is as follows:
Beef leach liquor 1L, peptone 10g, sodium-chlor 5g, aseptic defiber horse blood, sheep blood or rabbit blood 100ml, glucose 3g, hydrochloric acid casein hydrolysate 2g, pH7.0 ~ 7.2.
The present invention compared with prior art, has the following advantages and beneficial effect:
The dead bacterium of the A group meningitis cocci that the present invention cultivates is less, and the polysaccharide purification extracted is more prone to; And the extraction yield of polysaccharide in pod membrane is higher.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
The cultural method of A group meningitis cocci, comprises the following steps:
(1) the working seed lots bacterial classification of purifying is prepared: by A group meningitis Neisseria gonorrhoeae strain inoculation on pure medium, 20h is cultivated under 38 DEG C of conditions, select stalwartness and inoculate on pure medium without the bacterial classification of living contaminants and carry out second incubation, 12h is cultivated again under 38 DEG C of conditions, select stalwartness and bacterial classification without living contaminants adds in germfree defatted milk, mixing freeze-drying is prepared and obtains the working seed lots bacterial classification of purifying.
(2) working seed lots bacterial classification is dissolved in sterilized water, is seeded on 10% SBA substratum, be put under the environment of 35 ~ 37 DEG C and cultivate after 16 ~ 20 hours, pick out stalwartness and without the bacterial classification of living contaminants;
Purifying seed after unlatching is inoculated in half comprehensive liquid nutrient medium and carries out activation culture; The condition of activation culture is temperature 35 ~ 37 DEG C, pH7.0 ~ 7.2, stirring velocity 140 ~ 150r/min, and incubation time is 6 ~ 8h;
Finally by the purifying seed after activation through three generations's amplification cultivation, the production seed that quantity is suitable can be prepared; Wherein amplification cultivation substratum is in half comprehensive liquid nutrient medium; Breeding condition is temperature 35 ~ 37 DEG C, pH7.0 ~ 7.2, stirring velocity 140 ~ 150r/min.
(3) production seed is seeded to fermentor cultivation 6 ~ 12 hours; In fermentor tank, substratum is half synthetic medium, and temperature is 35 ~ 37 DEG C, and medium pH is 7.0 ~ 7.2, and in fermentor tank, stirring velocity is 140 ~ 150r/min, and inoculum size is 10 ~ 150,000/ml.
In the present embodiment, the concrete breeding condition in each step is as follows:
The breeding condition that step (2) working seed lots bacterial classification is opened is 35 DEG C, cultivates 18 hours; The condition of activation culture is temperature 37 DEG C, pH7.2, stirring velocity 140r/min, and incubation time is 6 ~ 8h; The breeding condition of amplification cultivation is temperature 36 DEG C, pH7.0, stirring velocity 145r/min;
In step (3), the breeding condition of fermentor cultivation is temperature is 37 DEG C, and medium pH is 7.0, and stirring velocity is 145r/min, and inoculum size is 120,000/ml.
And the pure medium adopted in step (1) is for adding antibiotic chocolate culture-medium, effectively reaches the object of purifying; Opening working seed lots substratum that bacterial classification uses in step (2) is do not add antibiotic chocolate culture-medium, i.e. 10% SBA substratum, by effectively reaching the effect of opening working seed lots bacterial classification after the cultivation of this substratum.
It is all half comprehensive liquid nutrient mediums that substratum used is cultivated in activation and amplification, and the proportioning of this substratum is: beef leach liquor 1L, peptone 10g, sodium-chlor 5g, aseptic defiber horse blood, sheep blood or rabbit blood 100ml, glucose 3g, hydrochloric acid casein hydrolysate 2g, pH7.0 ~ 7.2.
Embodiment 2
The difference of the present embodiment and embodiment 1 is only, the concrete breeding condition in each step is different, specifically arranges as follows:
The breeding condition that step (2) working seed lots bacterial classification is opened is 36 DEG C, cultivates 16 hours; The condition of activation culture is temperature 37 DEG C, pH7.0, stirring velocity 145r/min, and incubation time is 8h; The breeding condition of amplification cultivation is temperature 37 DEG C, pH7.0, stirring velocity 150r/min;
In step (3), the breeding condition of fermentor cultivation is temperature is 37 DEG C, and medium pH is 7.0, and stirring velocity is 150r/min, and inoculum size is 150,000/ml.
Embodiment 3
The difference of the present embodiment and embodiment 1 is only, the concrete breeding condition in each step is different, specifically arranges as follows:
The breeding condition that step (2) working seed lots bacterial classification is opened is 37 DEG C, cultivates 18 hours; The condition of activation culture is temperature 36 DEG C, pH7.2, stirring velocity 150r/min, and incubation time is 7h; The breeding condition of amplification cultivation is temperature 37 DEG C, pH7.2, stirring velocity 145r/min;
In step (3), the breeding condition of fermentor cultivation is temperature is 37 DEG C, and medium pH is 7.2, and stirring velocity is 145r/min, and inoculum size is 100,000/ml.
Above-described embodiment is only the preferred embodiments of the present invention, not limiting the scope of the invention, as long as adopt principle of design of the present invention, and the change carried out non-creativeness work on this basis and make, all should belong within protection scope of the present invention.

Claims (6)

  1. The cultural method of 1.A group meningitis cocci, is characterized in that, comprises the following steps:
    (1) the working seed lots bacterial classification of purifying is prepared;
    (2) open working seed lots bacterial classification, be seeded in half synthetic medium, cultivate 16 ~ 24 hours under temperature 35 ~ 37 DEG C of conditions, then through three generations's amplification cultivation, the production seed that preparation quantity is suitable;
    (3) production seed is seeded to fermentor cultivation 6 ~ 12 hours; In fermentor tank, substratum is half synthetic medium, and temperature is 35 ~ 37 DEG C, and medium pH is 7.0 ~ 7.2, and in fermentor tank, stirring velocity is 140 ~ 150r/min, and inoculum size is 10 ~ 150,000/ml.
  2. 2. the cultural method of A group meningitis cocci according to claim 1, is characterized in that, the detailed process of described step (1) is as follows:
    By A group meningitis Neisseria gonorrhoeae strain inoculation on pure medium, 20h is cultivated under 38 DEG C of conditions, select stalwartness and inoculate on pure medium without the bacterial classification of living contaminants and carry out second incubation, 12h is cultivated again under 38 DEG C of conditions, select stalwartness and bacterial classification without living contaminants adds in germfree defatted milk, mixing freeze-drying is prepared and obtains the working seed lots bacterial classification of purifying.
  3. 3. the cultural method of A group meningitis cocci according to claim 1, is characterized in that, the opening process of described purifying seed is as follows:
    Working seed lots bacterial classification is dissolved in sterilized water, is seeded on 10% SBA substratum, be put under the environment of 35 ~ 37 DEG C and cultivate after 16 ~ 20 hours, pick out stalwartness and without the bacterial classification of living contaminants.
  4. 4. the cultural method of A group meningitis cocci according to claim 3, is characterized in that, the concrete cultivating process of described step (2) is as follows:
    Purifying seed after unlatching is inoculated in half comprehensive liquid nutrient medium and carries out activation culture; The condition of activation culture is temperature 35 ~ 37 DEG C, pH7.0 ~ 7.2, stirring velocity 140 ~ 150r/min, and incubation time is 6 ~ 8h; Finally by the purifying seed after activation through three generations's amplification cultivation, the production seed that quantity is suitable can be prepared.
  5. 5. the cultural method of A group meningitis cocci according to claim 4, is characterized in that, the condition of described three generations's amplification cultivation is: amplification cultivation substratum is in half comprehensive liquid nutrient medium; Breeding condition is temperature 35 ~ 37 DEG C, pH7.0 ~ 7.2, stirring velocity 140 ~ 150r/min.
  6. 6. the cultural method of A group meningitis cocci according to claim 1, is characterized in that, the composition and ratio of described half comprehensive liquid nutrient medium is as follows:
    Beef leach liquor 1L, peptone 10g, sodium-chlor 5g, aseptic defiber horse blood, sheep blood or rabbit blood 100ml, glucose 3g, hydrochloric acid casein hydrolysate 2g, pH7.0 ~ 7.2.
CN201510543425.1A 2015-08-31 2015-08-31 A-group Neisseria meningitidis cultivation method Pending CN105039227A (en)

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PCT/CN2016/078086 WO2017036140A1 (en) 2015-08-31 2016-03-31 A-group neisseria meningitidis cultivation method

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017036140A1 (en) * 2015-08-31 2017-03-09 成都欧林生物科技股份有限公司 A-group neisseria meningitidis cultivation method
CN108690816A (en) * 2017-04-12 2018-10-23 成都生物制品研究所有限责任公司 A kind of the non-animal source culture medium and its cultural method of A groups of neisseria meningitis inflammation coccus

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102327605B (en) * 2011-08-25 2013-01-30 成都康华生物制品有限公司 Preparation process of meningococcal polysaccharide vaccine
CN103721249B (en) * 2014-01-16 2015-06-24 华兰生物工程股份有限公司 Meningitis vaccine and preparation method thereof
CN104689309A (en) * 2015-03-27 2015-06-10 成都欧林生物科技股份有限公司 Separated and purified acellular pertussis-diphtheria-tetanus, b-type haemophilus influenzae and A-group and C-group meningococcus combined vaccine and preparation method thereof
CN105039227A (en) * 2015-08-31 2015-11-11 成都欧林生物科技股份有限公司 A-group Neisseria meningitidis cultivation method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017036140A1 (en) * 2015-08-31 2017-03-09 成都欧林生物科技股份有限公司 A-group neisseria meningitidis cultivation method
CN108690816A (en) * 2017-04-12 2018-10-23 成都生物制品研究所有限责任公司 A kind of the non-animal source culture medium and its cultural method of A groups of neisseria meningitis inflammation coccus

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Application publication date: 20151111