CN115478034A - Meningococcus fermentation culture method, fermentation liquid, refined polysaccharide and application - Google Patents
Meningococcus fermentation culture method, fermentation liquid, refined polysaccharide and application Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 86
- 230000004151 fermentation Effects 0.000 title claims abstract description 86
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 31
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 31
- 150000004676 glycans Chemical class 0.000 title claims abstract description 30
- 238000012136 culture method Methods 0.000 title claims abstract description 25
- 239000001963 growth medium Substances 0.000 claims abstract description 72
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- 230000001580 bacterial effect Effects 0.000 claims abstract description 45
- 201000009906 Meningitis Diseases 0.000 claims abstract description 25
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- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 20
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- 239000012362 glacial acetic acid Substances 0.000 claims description 31
- 238000012807 shake-flask culturing Methods 0.000 claims description 16
- 241001478240 Coccus Species 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 9
- 108010060123 Conjugate Vaccines Proteins 0.000 claims description 2
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 229940031670 conjugate vaccine Drugs 0.000 claims description 2
- 229940031937 polysaccharide vaccine Drugs 0.000 claims description 2
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- 229960005486 vaccine Drugs 0.000 abstract description 4
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- 239000002244 precipitate Substances 0.000 description 9
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
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- 239000008215 water for injection Substances 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 5
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- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
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- 229910000403 monosodium phosphate Inorganic materials 0.000 description 4
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- 239000000843 powder Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 4
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- 239000012138 yeast extract Substances 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 3
- 235000019393 L-cystine Nutrition 0.000 description 3
- 239000004158 L-cystine Substances 0.000 description 3
- -1 L-sodium glutamate Substances 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 239000006161 blood agar Substances 0.000 description 3
- 229960003067 cystine Drugs 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 208000006400 Arbovirus Encephalitis Diseases 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 206010052369 Encephalitis lethargica Diseases 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 206010027249 Meningitis meningococcal Diseases 0.000 description 1
- 201000010924 Meningococcal meningitis Diseases 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
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- 238000003756 stirring Methods 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 201000002498 viral encephalitis Diseases 0.000 description 1
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- 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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Abstract
The application relates to the field of bioengineering, in particular to a meningococcus fermentation culture method, fermentation liquor, refined polysaccharide and application. A meningococcal fermentation culture method comprises the following steps: s1, starting strains in liquid recovery culture, inoculating the strains to a meningitis coccal culture medium, and inoculating the strains to OD 600 When the culture reaches 0.6-1.0, finishing the culture; s2, liquid shake culture, namely inoculating a generation of liquid culture bacterium liquid into a meningitis coccal culture medium, wherein OD is obtained 600 When the culture reaches 1.0-2.0, finishing the culture; s3, inoculating the second-generation liquid culture bacterium liquid to meningococcus by fermentation cultureFermenting in a culture medium, controlling the fermentation environment to be faintly acid, controlling the dissolved oxygen to be 20% -40%, and ending the culture when the strain grows into a stable period. According to the application, the introduction of the sheep blood component in the semi-integrated culture medium is reduced through a liquid resuscitation passage mode, and the safety of the vaccine is improved; meanwhile, the fermentation culture time is reduced, and the concentration of the subculture bacterial liquid is convenient to monitor and control.
Description
Technical Field
The application relates to the field of bioengineering, in particular to a meningococcal fermentation culture method, fermentation liquid, refined polysaccharide and application.
Background
Meningococcal meningitis (meningitis), also known as epidemic cerebrospinal meningitis (NM), is called epidemic cerebrospinal meningitis for short, and is a respiratory infectious disease seriously harming human health caused by neisseria meningitis.
Meningococci are divided into several groups, depending on the chemical and serological differences of the capsular polysaccharide, of which group A, C, Y, W is a common pathogenic bacterium. Epidemic prevention of epidemic encephalitis remains one of the important topics in the prevention and treatment of childhood infectious diseases in the world today.
At present, a semi-integrated culture medium is adopted as a solid resuscitation passage mode, and the defects of the semi-integrated culture medium are that (1) the first-generation solid semi-integrated culture medium in the prior art contains sheep blood, animal-derived components are introduced, and the semi-integrated culture medium is not necessarily advocated and is not used as much as possible from the safety of vaccines; (2) the solid culture takes 8-16 hours generally; (3) the end point of solid culture, unlike liquid culture, can monitor OD value in real time, thus being inconvenient to control the concentration of subculture bacteria.
In addition, how to continuously break through and improve the yield and reduce the production cost on the basis of the prior art is also continuously pursued by the vaccine industry. Therefore, a higher-yield meningococcal fermentation culture method is needed.
Disclosure of Invention
In order to improve the polysaccharide yield of meningococcal capsules, the application provides a meningococcal fermentation culture method.
The application provides a meningococcus fermentation culture method, which adopts the following technical scheme:
in a first aspect, the present application provides a meningococcal fermentation culture process comprising the steps of:
s1, liquid recovery culture
Starting the strain, inoculating to the culture medium of meningococcus, OD 600 When the concentration reaches 0.6-1.0, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating a generation of liquid culture bacterial liquid into a culture medium of meningococcus, and inoculating OD 600 And finishing the culture when the concentration reaches 1.0-2.0 to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus for fermentation, controlling the fermentation environment to be faintly acid, controlling the dissolved oxygen to be 20% -40%, and ending the culture when the bacterial grows into a stable period.
By adopting the scheme: by controlling the inoculation proportion of the subculture mode and combining the special fermentation culture mode, the application can enable meningococcus to produce seed liquid with higher capsular polysaccharide content in a weak acid environment when the meningococcus is cultured in a liquid shake flask. And the meningococcus is controlled in a specific culture environment in the fermentation process, so that more capsular polysaccharide is formed by fermentation, and the yield of polysaccharide is improved.
Optionally, the culture conditions in step S1 and/or S2 include a temperature of 37 + -1.5 deg.C and a rotation speed of 200-400 rpm for 3-12 hours.
Optionally, the culturing conditions in step S1 and/or S2 include controlling the culture environment to be acidic.
Optionally, the culture pH is controlled to 6.5-6.8.
Optionally, in step S3, the second-generation liquid culture broth is inoculated into a culture medium for meningococcus for fermentation, pH is controlled to 6.8-7.4, after culturing for 3-12 hours, pH is controlled to 6.3-7.0, dissolved oxygen is controlled to 20% -40%, the strain grows into a stable period, and the culture is ended.
In a second aspect, the present application provides a meningococcal fermentation broth prepared by the above fermentation culture method.
In a third aspect, the present application provides a refined polysaccharide prepared from a meningococcal fermentation broth.
In a fourth aspect, the present application provides a meningococcal polysaccharide vaccine or conjugate vaccine prepared from a meningococcal refined polysaccharide.
In conclusion, the invention has the following beneficial effects:
1. directly through liquid passage, on one hand, the first generation culture time is reduced, the fermentation production efficiency is improved, and on the other hand, the introduction of animal-derived components in a culture medium is reduced, so that the safety of the vaccine is improved.
2. This application can even monitor OD value through whole liquid culture to be convenient for control the concentration of passage fungus liquid, make fungus liquid produce and results in more suitable environment.
3. By controlling the inoculation proportion of the subculture mode and combining the special fermentation culture mode, the application can enable meningococcus to produce seed liquid with higher capsular polysaccharide content in a weak acid environment when the meningococcus is cultured in a liquid shake flask. And in the fermentation process, the meningococcus is controlled in a specific culture environment, so that more capsular polysaccharide is formed by fermentation, and the yield of polysaccharide is improved.
Detailed Description
Raw materials and sources
All types of meningitis strains are purchased from China medical culture Collection of microorganisms and cell culture Collection (CMCC). Neisseria meningitidis group A4CMCC29201, neisseria meningitidis group C11CMCC29205, neisseria meningitidis group Y strain YCMCC29303, neisseria meningitidis group W135CMCC29055.
Preparation of culture Medium for meningococcus
Preparation example 1
The culture medium for meningitis cocci is prepared from solution A and solution B according to the volume ratio of 9:1. When the preparation method is specifically used, each liter of the meningitis coccus culture medium comprises 900mL of the solution A and 100mL of the solution B. Wherein, every liter of the solution A comprises: 6g of casein, 4g of yeast extract powder, 0.125g of amino acid nutrient, 0.2g of monopotassium phosphate, 0.99g of sodium dihydrogen phosphate, 1.0g L-sodium glutamate, 0.75g of ammonium chloride and 3g of sodium chloride; wherein the amino acid nutrient is prepared from 0.025g L-cystine and 0.1g glycine.
Each liter of the B liquid comprises: 0.35g magnesium sulfate, 0.005g manganese sulfate monohydrate, 10g glucose.
The preparation method of the meningococcus culture medium comprises the following steps:
(1) Weighing acid hydrolyzed casein, yeast extract powder, L-cystine, potassium dihydrogen phosphate, sodium dihydrogen phosphate, glycine, L-sodium glutamate, ammonium chloride and sodium chloride according to the preparation amount, adding water for injection to dissolve, and adjusting pH value to 7.3 with 8% sodium hydroxide solution to obtain solution A;
(2) Weighing magnesium sulfate, manganese sulfate monohydrate and glucose according to the preparation amount, and adding water for injection to dissolve to obtain a solution B;
(3) Sterilizing the solution A and the solution B in vacuum sterilizing cabinet at 115 deg.C for 30min; and mixing the solution A and the solution B in proportion after sterilization to obtain the culture medium for the meningococcus.
It should be noted that, the content and ratio of the raw material formula can be adjusted adaptively according to actual situations, for example, the culture medium of meningococcus can be, but is not limited to: is prepared from liquid A and liquid B according to the volume ratio of (8.5-9.5) to (0.5-1.5).
The raw material ratio of each liter of the solution A can be but is not limited to: 5-8g of casein, 3-5g of yeast extract powder, 0.1-0.15g of amino acid nutrient, 0.15-0.25g of monopotassium phosphate, 0.98-1.0g of sodium dihydrogen phosphate, 0.5-1.0g of g L-sodium glutamate, 0.5-1.0g of ammonium chloride and 2-4g of sodium chloride; the pH is adjusted to 7.2-7.4, and other strong or weak bases can be adopted.
The raw material ratio of each liter of the solution B can be but is not limited to: 0.3-0.4g of magnesium sulfate, 0.004-0.006g of manganese sulfate monohydrate and 9-11g of glucose.
It is understood that the preparation conditions can be adjusted according to the actual situation, for example, the sterilization temperature is 110-120 ℃, and the sterilization time is 20-40min.
Preparation example 2
The difference from the preparation example 1 is that the preparation method of the culture medium for the meningitis coccus comprises the following steps:
(1) Weighing acid hydrolyzed casein, yeast extract powder, L-cystine, potassium dihydrogen phosphate, sodium dihydrogen phosphate, glycine, L-sodium glutamate, ammonium chloride and sodium chloride according to the preparation amount, adding water for injection to dissolve, and adjusting pH value to 7.3 with 8% sodium hydroxide solution to obtain solution A;
(2) Weighing magnesium sulfate, manganese sulfate monohydrate and glucose according to the preparation amount, and adding water for injection to dissolve to obtain a solution B;
(3) Mixing solution A and solution B at a certain proportion, sterilizing in a vacuum sterilization cabinet at 115 deg.C for 30min to obtain culture medium for meningococcus.
Examples
Example 1 provides a meningococcal fermentation culture method, comprising:
s1, liquid recovery culture
A, C, Y and W135 strain were inoculated to the culture medium of meningococcus of preparation example 1 at 37 + -1.5 deg.C, pH was controlled to 6.5 with glacial acetic acid, and the culture was performed at 200rpm for 3 hr, OD 600 When the concentration reached 0.6, the culture was terminated to obtain a first-generation liquid culture medium.
S2, liquid shake flask culture
Inoculating the first generation liquid culture liquid into the culture medium of meningococcus provided by preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.5 with glacial acetic acid, culturing at 200rpm for 3 hr, and culturing at OD 600 When the concentration reaches 1.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second generation liquid culture bacteria liquid into a culture medium of the meningitis cocci for fermentation, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 2
The meningococcal fermentation culture method provided in embodiment 2 includes the following steps:
s1, liquid recovery culture
A, C, Y and W135 strain were inoculated to the culture medium of meningococcus of preparation example 1 at 37 + -1.5 deg.C, pH of the culture was controlled to 6.6 with glacial acetic acid, and the culture was performed at 200rpm for 3 hr, OD 600 When the concentration reaches 0.6, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating the first generation liquid culture liquid into the culture medium of meningococcus provided by preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.6 with glacial acetic acid, culturing at 200rpm for 3 hr, and culturing at OD 600 When the concentration reaches 1.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus for fermentation, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 3
Example 3 provides a meningococcal fermentation culture method, comprising:
s1, liquid recovery culture
A, C, Y and W135 strain were inoculated to the culture medium of meningococcus of preparation example 1 at 37 + -1.5 deg.C, pH of the culture was controlled to 6.8 with glacial acetic acid, and the culture was performed at 200rpm for 3 hr, OD 600 When the concentration reached 0.6, the culture was terminated to obtain a first-generation liquid culture medium.
S2, liquid shake flask culture
The first generation liquid culture broth was inoculated into the culture medium for meningococcus provided in preparation example 1 at a culture temperature of 37. + -. 1.5 ℃ and pH of 6.8 with glacial acetic acid, and cultured at 200rpm for 3 hours at OD 600 When the concentration reaches 1.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus for fermentation, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 4
Example 4 provides a meningococcal fermentation culture method, including the following steps:
s1, liquid recovery culture
A, C, Y and W135 strain were inoculated to the culture medium of meningococcus of preparation example 1 at 37 + -1.5 deg.C, pH of the culture was controlled to 6.2 with glacial acetic acid, and the culture was performed at 200rpm for 3 hr, OD 600 When the concentration reaches 0.6, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating the first generation liquid culture broth into culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C while controlling pH to 6.8 with glacial acetic acid, culturing at 200rpm for 3 hr,OD 600 when the concentration reaches 1.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus for fermentation, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 5
Example 5 provides a meningococcal fermentation culture method, comprising:
s1, liquid recovery culture
A, C, Y and W135 strain were inoculated to the culture medium of meningococcus of preparation example 1 at 37 + -1.5 deg.C, pH of the culture was controlled to 6.2 with glacial acetic acid, and the culture was performed at 200rpm for 3 hr, OD 600 When the concentration reaches 0.6, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
The first generation liquid culture broth was inoculated into the culture medium for meningococcus provided in preparation example 1 at a culture temperature of 37. + -. 1.5 ℃ and pH of 6.9 with glacial acetic acid, and cultured at 200rpm for 3 hours at OD 600 When the concentration reaches 1.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus for fermentation, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 6
The meningococcal fermentation culture method provided in embodiment 6 includes the following steps:
s1, liquid recovery culture
A, C, Y and W135 strain were inoculated to the culture medium of meningococcus of preparation example 1 at 37 + -1.5 deg.C, pH of the culture was controlled to 6.6 with glacial acetic acid, and the culture was performed at 200rpm for 4 hr, OD 600 When the concentration reaches 0.8, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating the first generation liquid culture solution into the culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.6 with glacial acetic acid, culturing at 250rpm for 4 hr, and culturing at OD 600 When the concentration reaches 1.5, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus for fermentation, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 7
Example 7 provides a meningococcal fermentation culture method, comprising:
s1, liquid recovery culture
Opening A, C, Y and W135 strain, inoculating to the culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C under pH control of glacial acetic acid at 300rpm for 5 hr, and culturing at OD 600 When the concentration reaches 1.0, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating the first generation liquid culture liquid into the culture medium of meningococcus provided by preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.6 with glacial acetic acid, culturing at 400rpm for 5 hr, and culturing at OD 600 When the culture reaches 2.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second generation liquid culture bacteria liquid into a culture medium of the meningitis cocci for fermentation, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 8
The meningococcal fermentation culture method provided in embodiment 8 includes the following steps:
s1, liquid recovery culture
A, C, Y and W135 strain were separately inoculated into the culture medium of meningococcus provided in preparation example 1 at 37 + -1.5 deg.C, pH was controlled to 6.6 with glacial acetic acid, and the culture was performed at 300rpm5 hours, OD 600 When the concentration reached 1.0, the culture was terminated to obtain a first-generation liquid culture medium.
S2, liquid shake flask culture
Inoculating the first generation liquid culture solution into the culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.6 with glacial acetic acid, culturing at 400rpm for 5 hr, and culturing at OD 600 When the concentration reaches 2.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus for fermentation, controlling the dissolved oxygen to be 30%, and ending the culture when the strain grows into a stable period.
Example 9
Example 9 provides a meningococcal fermentation culture method, comprising:
s1, liquid recovery culture
A, C, Y and W135 strain were inoculated to the culture medium of meningococcus of preparation example 1 at 37 + -1.5 deg.C, pH was controlled to 6.6 with glacial acetic acid, and the culture was performed at 300rpm for 5 hr, OD 600 When the concentration reaches 1.0, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating the first generation liquid culture solution into the culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.6 with glacial acetic acid, culturing at 400rpm for 5 hr, and culturing at OD 600 When the concentration reaches 2.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus for fermentation, controlling the dissolved oxygen to be 40%, and ending the culture when the strain grows into a stable period.
Example 10
Example 10 provides a meningococcal fermentation culture method, comprising:
s1, liquid recovery culture
Are separately openedStarting A, C, Y and W135 group strains, respectively inoculating to the culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C with glacial acetic acid to control pH to 6.6, culturing at 300rpm for 5 hr, and culturing at OD 600 When the concentration reaches 1.0, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating the first generation liquid culture solution into the culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.6 with glacial acetic acid, culturing at 400rpm for 5 hr, and culturing at OD 600 When the concentration reaches 2.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus, culturing at the temperature of 37 +/-1.5 ℃, at the culture pH of 6.8, and culturing for 3 hours at the rotating speed of 100 rpm;
after fermenting for 3h, controlling the culture pH to 6.6 by adopting glacial acetic acid, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 11
Example 11 provides a meningococcal fermentation culture method, comprising:
s1, liquid recovery culture
Opening A, C, Y and W135 strain, inoculating to the culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C under pH control of glacial acetic acid at 300rpm for 5 hr, and culturing at OD 600 When the concentration reaches 1.0, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating the first generation liquid culture solution into the culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.6 with glacial acetic acid, culturing at 400rpm for 5 hr, and culturing at OD 600 When the concentration reaches 2.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus, culturing at the temperature of 37 +/-1.5 ℃, at the culture pH of 7.3, and culturing for 3 hours at the rotating speed of 100 rpm;
after fermenting for 3h, controlling the culture pH to 6.6 by adopting glacial acetic acid, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 12
Example 12 provides a meningococcal fermentation culture method, comprising:
s1, liquid recovery culture
A, C, Y and W135 strain were inoculated to the culture medium of meningococcus of preparation example 1 at 37 + -1.5 deg.C, pH was controlled to 6.6 with glacial acetic acid, and the culture was performed at 300rpm for 5 hr, OD 600 When the concentration reaches 1.0, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating the first generation liquid culture solution into the culture medium of meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.6 with glacial acetic acid, culturing at 400rpm for 5 hr, and culturing at OD 600 When the culture reaches 2.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus, culturing at the temperature of 37 +/-1.5 ℃, at the culture pH of 7.4, and culturing for 3 hours at the rotating speed of 100 rpm;
after fermenting for 3h, controlling the culture pH to 7.0 by adopting glacial acetic acid, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 13
Example 13 provides a meningococcal fermentation culture method, comprising:
s1, liquid recovery culture
A, C, Y and W135 strain were inoculated to the culture medium of meningococcus of preparation example 1 at 37 + -1.5 deg.C, pH was controlled to 6.6 with glacial acetic acid, and the culture was performed at 300rpm for 5 hr, OD 600 When the concentration reaches 1.0, the culture is finished to obtain a first generation liquid culture bacterial liquid.
S2, liquid shake flask culture
Inoculating the first generation liquid culture liquid into the culture medium of meningococcus provided by preparation example 1, culturing at 37 + -1.5 deg.C, controlling pH to 6.6 with glacial acetic acid, culturing at 400rpm for 5 hr, and culturing at OD 600 When the concentration reaches 2.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
Inoculating the second generation liquid culture bacterial liquid into a culture medium of the meningococcus, culturing at the temperature of 37 +/-1.5 ℃, with the culture pH of 6.9, and culturing for 3 hours at the rotating speed of 300 rpm;
after fermenting for 3h, controlling the culture pH to 6.4 by adopting glacial acetic acid, controlling the dissolved oxygen to be 20%, and ending the culture when the strain grows into a stable period.
Example 14
The difference from example 10 is that the culture medium for meningococcus provided in preparation example 2 was used in both step S1 and step S2.
Comparative example 1
The difference from example 5 is that OD in step S1 600 When the concentration reached 0.3, the culture was terminated.
Comparative example 2
The difference from example 5 is that OD in step S1 600 When 1.4 was reached, the culture was terminated.
Comparative example 3
The difference from example 5 is that in step S2, OD is 600 When the concentration reached 0.8, the culture was terminated.
Comparative example 4
The difference from example 5 is that in step S2, OD is 600 When the concentration reached 2.3, the culture was terminated.
Comparative example 5
The difference from example 5 is that in step S3, the dissolved oxygen is 15%.
Comparative example 6
The difference from example 5 is that in step S3, the dissolved oxygen was 50%.
Comparative example 7
The difference from example 5 is that the meningococcal fermentation culture method is different, and in particular,
the meningococcus fermentation culture method provided by the comparative example 7 comprises the following steps:
s1, solid recovery culture
Scraping A, C, Y and W135 meningococcal solid first generations with strain rod, inoculating to 10% sheep blood agar medium, smearing, placing the inoculated 10% sheep blood agar medium at 37 deg.C, and 7.5% CO 2 Culturing for 12 hours in the incubator;
s2, liquid shake flask culture
Sucking the culture medium prepared in the S1 by using a pipette, and rinsing the lawn on the 10% sheep blood agar culture medium; inoculating the strain in the culture medium prepared in the S1; the inoculated flask was placed in a large-volume shaker and incubated at 37 ℃ and 200rpm for 6 hours.
S3, fermentation culture
Inoculating the second generation liquid culture bacterial liquid into a meningococcal culture medium, culturing at the temperature of 37 +/-1.5 ℃ and the culture pH of 6.9, and culturing at the rotating speed of 300rpm until the strain grows into a stable stage, and ending the culture to obtain meningococcal fermentation liquid.
Performance detection
1. Effective yield test of refined polysaccharide:
a formaldehyde solution is added into the meningococcal fermentation liquid according to 1% (v/v) in the previous examples and comparative examples respectively, the meningococcal fermentation liquid is sterilized for 60 minutes, and then the meningococcal fermentation liquid is centrifuged by a centrifuge at 4000rpm and 10 ℃ for 15 minutes to remove thalli and collect supernatant. Adding 0.1% hexadecyl trimethyl ammonium bromide into the supernatant, mixing to form precipitate, and standing for 3 hr.
Centrifuging the supernatant at 4000rpm in a centrifuge at 10 deg.C for 15min, and collecting precipitate. Adding 2mol/L calcium chloride solution into the precipitate according to 4.0ml/g polysaccharide complex precipitate for dissociation, then adding water for injection (sterilization) to make the final concentration of calcium chloride lmol/L, and stirring for 1 hour to make the polysaccharide hexadecyl trimethyl ammonium bromide complex dissociate. Adding a pre-cooled 95% ethanol solution at the temperature of 2-8 ℃ until the final concentration is 25% (ml/m 1), uniformly mixing, and standing in a cold storage at the temperature of 2-8 ℃ for 3 hours. Centrifuging and collecting supernatant: the precipitate was centrifuged at 9000rpm for 40 minutes, and the supernatant was collected.
The supernatant was clarified and filtered through a 0.22 μm filter. Adding a pre-cooled 95% ethanol solution at the temperature of 2-8 ℃ into the supernatant until the final concentration is as follows: 75% (ml/m 1). After sufficient shaking, standing for 1-2 hours or overnight. The precipitate was collected by centrifugation at 4000rpm for 15 minutes, washed twice with 2-8 ℃ pre-cooled absolute ethanol (4000rpm, 15 minutes for each washing, and centrifugation at 15 ℃ or lower) in a volume of 2 times that of the polysaccharide, and washed twice with 2-8 ℃ pre-cooled acetone (4000rpm, 15 minutes for each washing, and centrifugation at 15 ℃ or lower) in a volume of 2 times that of the polysaccharide. Discarding supernatant, collecting polysaccharide precipitate, vacuum drying or blow drying to obtain crude polysaccharide product
The crude polysaccharide product is dissolved in 10% saturated neutral sodium acetate solution to make A, C, Y and W135 group concentration reach 10-15 mg/ml, and extracted 3-12 times by 10% saturated neutral sodium acetate cold phenol solution with 2 times polysaccharide volume. The supernatant was collected by centrifugation at 9000rpm for 40 minutes. Adding 8-15 times volume of injection water into the supernatant, and performing ultrafiltration to the original volume by adopting a 100KD tangential flow ultrafiltration system; then using 10-20 times volume of water for injection to carry out ultrafiltration dialysis (equal volume ultrafiltration) until the volume is the original volume; 2mol/L calcium chloride solution is added to a final concentration of 0.1mol/L. Adding pre-cooled 95% ethanol at 2-8 ℃ until the final concentration is: 75% (ml/m 1). After sufficient shaking, the mixture was allowed to stand for 2 hours, centrifuged at 4000rpm for 15 minutes to collect precipitates, washed twice with 2-8 ℃ pre-cooled absolute ethanol (4000rpm, 15 minutes per washing, centrifuged at 15 ℃ or lower) in a volume 2 times that of the polysaccharide (and washed twice with 2-8 ℃ pre-cooled acetone (4000rpm, 15 minutes per washing, centrifuged at 15 ℃ or lower) in a volume 2 times that of the polysaccharide). And (4) discarding the supernatant, collecting polysaccharide precipitate, and vacuum drying to obtain the refined polysaccharide.
The polysaccharide yields of the above examples and comparative examples were weighed respectively, and the results are shown in Table 1.
TABLE 1 Fine polysaccharide yield Table
The above embodiments are preferred embodiments of the present application, and the protection scope of the present application is not limited by the above embodiments, so: all equivalent changes made according to the structure, shape and principle of the present application shall be covered by the protection scope of the present application.
Claims (10)
1. A meningococcus fermentation culture method is characterized in that: the method comprises the following steps:
s1, liquid recovery culture
Starting the strain, inoculating to the culture medium of meningococcus, OD 600 When the concentration reaches 0.6-1.0, finishing the culture to obtain a first generation liquid culture bacterial liquid;
s2, liquid shake flask culture
Inoculating a generation of liquid culture bacteria liquid into a culture medium of meningococcus, OD 600 When the concentration reaches 1.0-2.0, finishing the culture to obtain a second-generation liquid culture bacterial liquid;
s3, fermentation culture
Inoculating the second-generation liquid culture bacterial liquid into a culture medium of the meningitis coccus for fermentation, controlling the fermentation environment to be faintly acid, controlling the dissolved oxygen to be 20% -40%, and ending the culture when the bacterial grows into a stable period.
2. The meningococcal fermentation culture process of claim 1, wherein: the culture conditions in step S1 and/or S2 include a temperature of 37 + -3.5 deg.C and a rotation speed of 200-400 rpm for 3-12 hours.
3. The meningococcal fermentation culture process of claim 1 or 2, wherein: the culture conditions in step S1 and/or S2 include controlling the culture environment to be acidic.
4. The meningococcal fermentation culture process of claim 3, wherein: controlling the culture pH value to be 6.5-6.8.
5. The meningococcal fermentation culture process of claim 1, wherein: and step S3, inoculating the second-generation liquid culture bacterium liquid into a culture medium of the meningitis coccus for fermentation, controlling the pH to be 6.8-7.4, controlling the pH to be 6.3-7.0 and the dissolved oxygen to be 18% -44% after the culture is carried out for 3-12 h, and ending the culture when the strain grows to enter a stable period.
6. The meningococcal fermentation culture process of claim 4 or 5, wherein: the pH is controlled using a weak acid.
7. The meningococcal fermentation culture process of claim 6, wherein: the weak acid is glacial acetic acid.
8. A meningococcal fermentation broth produced by the fermentation culture process of any one of claims 1 to 7.
9. A purified polysaccharide produced from the meningococcal fermentation broth of claim 8.
10. A meningococcal polysaccharide vaccine or conjugate vaccine prepared from the meningococcal refined polysaccharide of claim 9.
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