CN107723268A - Bacterial screening method for pneumonia capsular polysaccharide large-scale production - Google Patents
Bacterial screening method for pneumonia capsular polysaccharide large-scale production Download PDFInfo
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- CN107723268A CN107723268A CN201711235794.XA CN201711235794A CN107723268A CN 107723268 A CN107723268 A CN 107723268A CN 201711235794 A CN201711235794 A CN 201711235794A CN 107723268 A CN107723268 A CN 107723268A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
Abstract
The invention discloses the bacterial screening method for pneumonia capsular polysaccharide large-scale production, comprise the following steps：Step 1, fluid nutrient medium is prepared；Step 2, solid medium is prepared；Step 3, the fluid nutrient medium prepared using step 1 is carried out culture and obtains generation strain；Step 4, the solid medium prepared using step 2 is carried out culture and obtains second-generation bacterial kind；Step 5, the liquid base culture prepared using step 1, three generations's strain is obtained；Step 6, capsule stain, Gram's staining, the bacterium solution of the thick strain reference numeral of pod membrane is retained；Step 7, four be commissioned to train it is foster：The fluid nutrient medium culture that the thick bacterium solution of pod membrane is prepared in step 1；Step 8, strain harvest, preservation.The thicker strain of pod membrane is filtered out by mode provided by the invention, about 80% capsular polysaccharide yield is improved on the basis of in the past, and polysaccharide meets European Pharmacopoeia standard through examining, and substantially increases polysaccharide yield, improves production efficiency, reduces cost.
Description
Technical field
The present invention relates to microbial technology field, and in particular to the bacterial screening for pneumonia capsular polysaccharide large-scale production
Method.
Background technology
Streptococcus pneumonia (streptococcus pneumoniae) was isolated by Pasteur first in 1881, was great Ye
Property pneumonia, meningitis, one of the principal causative source of bronchitis, it can also cause tympanitis, nasosinusitis and bacteremia etc..Pneumonia
Streptococcal infection is one of major reason for causing death in the world.The U.S. has data to show, and estimation is annual 40~
500000 people suffer from an inflammation of the lungs coccus pneumonia, and case fatality rate is 5%~10%.In it can prevent the disease of death of child with vaccine, pneumonia
Disease ranks the first caused by streptococcus.With a large amount of uses of antibiotic, antibody-resistant bacterium is growing day by day, and medical field is paid close attention to again
The research and development of vaccine.Pneumococcus has now been found that 90 various serotypes.Its bacterium surface pod membrane has antigenicity, then by pneumonia ball
Granulose is covalently attached to carrier protein and makes thymus-dependent (TD) antigen, infant and old man can be produced anti-
Body, immune effect effectively improve.Existing vaccine is to extract its capsular polysaccharide to be made for raw material.
Since pneumovax is researched and developed, because its capsular polysaccharide yield is relatively low, only by expanding the scale of production as far as possible, with
Reach the purpose for improving polysaccharide yield, therefore production cost is higher.Improving polysaccharide yield can be from screening high-quality strain, optimization fermentation
The critical processes such as technique, optimized purification technique are set about.The thicker strain of pod membrane is screened, is one of critical process, from source
Solves this problem.
The content of the invention
The technical problems to be solved by the invention are that the capsular polysaccharide yield that medium culture streptococcus pneumonia obtains is relatively low,
Purpose is to provide the bacterial screening method for pneumonia capsular polysaccharide large-scale production, there is provided the culture medium of a kind of group of simplification
For the culture of strain, and suitable cultural method, the thicker strain of pod membrane is filtered out by mode provided by the invention,
About 80% capsular polysaccharide yield was improved on the basis of in the past, and polysaccharide meets European Pharmacopoeia standard through examining, and carries significantly
High polysaccharide yield, improves production efficiency, reduces cost.
The present invention is achieved through the following technical solutions：
For the bacterial screening method of pneumonia capsular polysaccharide large-scale production, comprise the following steps：
Step 1, fluid nutrient medium is prepared：Following raw material is weighed, above-mentioned raw materials are dissolved successively using water for injection,
Constant volume volume needed for will be carried out using the mixed solution after water for injection dissolving, and adjust pH value, liquid training is obtained after degerming
Support base：Solvent is that the relative usage of water for injection is 1000ml：
It is containing nitrogen source：Trypticase：15~80g, tryptone：20~70g, selected soya peptone：10~80g, soya peptone：10~
80g, acid hydrolyzed casein：10~70g, yeast extract：15~40g, yeast extract：Any three kinds of combinations in 5~40g
Thing；Carbon source is：Glucose：5.0~50g；Salt is：K2HPO4：0~12.0g, KCl：0~7.0g, Na2HCO3：0~5.0g,
NaCl：3.0~10.0g, MgSO4·7H2O：0.4~5.0g, CaCl2：0~0.14g；Trace element is：Cys hydrochloric acid
Salt：0.12~1.80g, nicotinic acid：0.5~1.2mg, adenine：5.5~10.5mg, FeSO4：2.5~6.5mg；
Step 2, solid medium is prepared：10~15g of agar is weighed, is dissolved in water for injection rear constant volume volume needed for
90%, the solution after dissolving carries out moist heat sterilization, standby after cooling；
Following raw material is weighed, is dissolved using water for injection, the mixed solution after dissolving is subjected to constant volume body needed for
Product, and pH value, bacteria removing are adjusted, it is eventually adding in agar medium, pours into plate, solid culture based solvent is made；For water
Relative usage be 1000ml：
It is trypticase containing nitrogen source：15~80g, tryptone：20~70g, selected soya peptone：10~80g, soya peptone：10~
80g, acid hydrolyzed casein：10~70g, yeast extract：15~40g, yeast extract：Any three kinds in 5~40g；Carbon source is
Glucose：5.0~50g；Salt is K2HPO4：0~12.0g, KCl：0~7.0g, Na2HCO3：0~5.0g, NaCl：3.0~
10.0g MgSO4·7H2O：0.4~5.0g, CaCl2：0~0.14g；Trace element is L-cysteine hydrochloride：0.12~
1.80g nicotinic acid：0.5~1.2mg, adenine：5.5~10.5mg, FeSO4：2.5~6.5mg；
Step 3, generation culture：Breakdown streptococcus pneumonia working seed lots strain, the Liquid Culture prepared with the step 1
After base dissolving fungus block, fluid nutrient medium 2~20ml, 35 DEG C~37 DEG C, CO prepared by inoculation step 12Throughput be 60%~
100%, 4~10h of quiescent culture, obtain generation strain；
Step 4, two be commissioned to train it is foster：Generation bacterium solution is transferred in solid medium plate prepared by step 2, separated with line
Method is inoculated with, 35 DEG C~37 DEG C, CO2Throughput is 60%~100%, 36~48h of quiescent culture, obtains second-generation bacterial kind；
Step 5, three generations cultivates：The single bacterium colony grown on picking solid medium 15~20, is inoculated in step 1 respectively
In 2~5ml of Liquid Culture of preparation, numbering, culture, cultivation temperature is 35 DEG C~37 DEG C, CO2Throughput is 60%~100%,
Incubation time is 8~17h, cultivate to bacterium solution it is substantially muddy when stop culture, obtain three generations's strain；
Step 6, capsule stain, Gram's staining：Each numbering is taken to correspond to bacterium solution after culture, with India ink staining, gentian violet
Or microscopy after the dyeing of husky of common dye dye liquor, the bacterium solution of the thick strain reference numeral of pod membrane is retained 1~5 group, the thin strain of remaining pod membrane
The bacterium solution of reference numeral is discarded；Repeat step 3, step 4 and step 5, again separation screening, the then pure culture filtered out；
Step 7, four be commissioned to train it is foster：The thick bacterium solution of pod membrane is gone to step to the fluid nutrient medium of 1 preparation by 10% inoculum concentration, carried out
Four generation amplification cultivations, cultivation temperature are 35 DEG C~37 DEG C, CO2Throughput is 60%~100%, and incubation time is 2~5h, treats bacterium
Liquid is obvious muddy, OD600 >=0.5 when harvest；
Step 8, strain harvest, preservation：Bacterium solution is centrifuged, bacterial sediment is collected and is stored in 10%~30% glycerine, point
Dress, is placed in liquid nitrogen and preserves.
Preferably, in the step 1, the raw material is added in same container, then to Sheng by raw mixture
Water for injection is added in container under constant agitation；After water for injection has been added, dissolved by heating, dissolve by heating step
Suddenly it is：30 DEG C first are warming up to by 25 DEG C with 1 DEG C/min heating rate, 10min is kept under 30 DEG C of temperature conditionss, then with 1
DEG C/min heating rate is warming up to 35 DEG C by 38 DEG C, keep 5min under 38 DEG C of temperature conditionss.
Preferably, in the step 1 and step 2, mass concentration is used to adjust pH value to 7.0 for 10% NaOH solution
~7.8.
Preferably, in the step 1, it is filtered under diminished pressure under aseptic technique through 0.1 μm of filtering degerming.
Preferably, in the step 2, water for injection is added into agar and is warming up to 95 DEG C and is dissolved；The step 2
It is middle by agar solution be cooled to 55~60 DEG C it is standby.
Preferably, in the step 2, moist heat sterilization concrete operations are：Moist heat sterilization temperature is 117~122 DEG C, and pressure is
0.09~0.12MPa, sterilization time are 15~30min.
Preferably, in the step 2, the bacteria removing with water for injection solution principle is：Pass through under aseptic technique
0.2 μm of filtering is filtered under diminished pressure degerming.
Preferably, the strain be used in streptococcus pneumonia 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A,
12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F totally 24 serotype.
The present invention compared with prior art, has the following advantages and advantages that the present invention is used for pneumonia capsular polysaccharide and advised
The bacterial screening method of modelling production,
The invention provides a kind of bacterial screening method for pneumonia capsular polysaccharide large-scale production, can be passed in strain
For when carry out good amplification cultivation, after fermented and cultured, its output of sugar is also higher；It is provided by the invention using this liquid and
The method of solid medium screening streptococcus pneumonia strain can substantially shorten the generation of screening and culturing, the bacterial activity after screening
Well, pod membrane is thicker, and bacterial concentration and polysaccharide yield all significantly improve.With simple to operate, quick, easy, reduction microbiological contamination of drawing materials
The advantages of risk.
Brief description of the drawings
Accompanying drawing described herein is used for providing further understanding the embodiment of the present invention, forms one of the application
Point, do not form the restriction to the embodiment of the present invention.In the accompanying drawings：
Fig. 1 is microscope oil mirror figure before screening；
Fig. 2 is microscope oil mirror figure after present invention screening.
Embodiment
For the object, technical solutions and advantages of the present invention are more clearly understood, with reference to embodiment, the present invention is made
Further to describe in detail, exemplary embodiment of the invention and its explanation are only used for explaining the present invention, are not intended as to this
The restriction of invention.
Embodiment 1~3
The invention provides a kind of Liquid Culture based formulas for streptococcus pneumonia, as shown in table 1：
The Liquid Culture based formulas of the streptococcus pneumonia of 1 embodiment of table 1~3
| Name of material | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| Nitrogen source 1 | Trypticase 15g | Tryptone 25g | Acid hydrolyzed casein 35g |
| Nitrogen source 2 | Soya peptone 20g | Selected soya peptone 40g | Selected soya peptone 45g |
| Nitrogen source 3 | Yeast extract 30g | Acid hydrolyzed casein 10g | Yeast extract 5g |
| Glucose | 10g | 22g | 31g |
| K2HPO4 | 10.0g | 4.3g | 7.5g |
| KCl | 3.4g | 5.2g | 1.8g |
| Na2HCO3 | 2.0g | 5.0g | 4.1g |
| NaCl | 4.0g | 6.5g | 9.0g |
| MgSO4·7H2O | 0.5g | 2.5g | 3.4g |
| CaCl2 | 0.02g | 0.09g | 0.12g |
| L-cysteine hydrochloride | 0.18g | 1.2g | 0.8g |
| Nicotinic acid | 0.6mg | 1.0mg | 0.8mg |
| Adenine | 6.0mg | 8.5mg | 10.0mg |
| FeSO4 | 3.6mg | 4.5mg | 5.7mg |
| pH | 7.2 | 7.5 | 7.7 |
Embodiment 4~6
The invention provides a kind of solid culture based formulas for streptococcus pneumonia, as shown in table 2：
The Liquid Culture based formulas of the streptococcus pneumonia of 2 embodiment of table 4~6
Embodiment 7
The invention provides a kind of method for screening streptococcus pneumonia, concretely comprise the following steps：
Step 1, strain is opened in liquid, generation culture：Breakdown streptococcus pneumonia working seed lots strain, with embodiment 1
After the fluid nutrient medium dissolving fungus block of offer, the fluid nutrient medium 20ml, 35 DEG C, CO of the offer of embodiment 1 are inoculated in2Throughput is
60%, quiescent culture 9h, obtain generation strain；
Step 2, solid line separation, two be commissioned to train it is foster：The solid medium that generation bacterium solution is transferred in the body of embodiment 4 is put down
In ware, it is inoculated with line partition method, 37 DEG C, CO2Throughput is 70%, quiescent culture 41h, obtains second-generation bacterial kind；
Step 3, liquid, three generations's culture are passed：The single bacterium colony grown on picking solid medium, it is inoculated in embodiment 1 and carries
Supply in 2ml Liquid Cultures；With method picking single bacterium colony, 20 are inoculated with fluid nutrient medium respectively, numbering, culture, and cultivation temperature is
35℃、CO2Throughput is 90%, incubation time 13h, cultivate to bacterium solution it is substantially muddy when stop culture, obtain three generations's strain；
4. capsule stain, Gram's staining：Each bacterium solution after numbering culture is taken, after being dyed with India ink staining (husky of common dye dye liquor)
Microscopy, the bacterium solution of the thick strain reference numeral of pod membrane is retained 2 groups, the bacterium solution of the thin strain reference numeral of remaining pod membrane is discarded；
5. pass liquid amplification, four be commissioned to train it is foster：The thick bacterium solution of pod membrane is turned into the fluid nutrient medium of embodiment 1 by 10% inoculum concentration,
Four generation amplification cultivations are carried out, cultivation temperature is 37 DEG C, CO2Throughput is 100%, incubation time 2.5h, and bacterium solution is bright during harvest
Aobvious muddy, OD600 0.73；
6. strain harvest, preserve：Bacterium solution is centrifuged, bacterial sediment is collected and is stored in 30% glycerine, dispense, be placed in liquid nitrogen
Middle preservation.
Embodiment 8：
The invention provides a kind of method for screening streptococcus pneumonia, concretely comprise the following steps：
Step 1, strain is opened in liquid, generation culture：Breakdown streptococcus pneumonia working seed lots strain, with embodiment 2
After fluid nutrient medium dissolving fungus block, fluid present invention culture medium 10ml, 36 DEG C, CO are inoculated in2Throughput is 80%, quiescent culture
8h, obtain generation strain；
Step 2, solid line separation, two be commissioned to train it is foster：The solid medium that generation bacterium solution is transferred in the body of embodiment 5 is put down
In ware, it is inoculated with line partition method, 36 DEG C, CO2Throughput is 60%, quiescent culture 38h, obtains second-generation bacterial kind；
Step 3, liquid, three generations's culture are passed：The single bacterium colony grown on picking solid medium, it is inoculated in embodiment 2 and carries
In the 5ml Liquid Cultures of confession；With method picking single bacterium colony, 15 are inoculated with fluid nutrient medium respectively, numbering, culture, and cultivation temperature is
36℃、CO2Throughput is 80%, incubation time 11h, cultivate to bacterium solution it is substantially muddy when stop culture, obtain three generations's strain；
Step 4, capsule stain, Gram's staining：Each bacterium solution after numbering culture is taken, with India ink staining (gentian violet solution)
Microscopy after dyeing, the bacterium solution of the thick strain reference numeral of pod membrane is retained 1 group, the bacterium solution of the thin strain reference numeral of remaining pod membrane
It is discarded；
Step 5, pass liquid amplification, four be commissioned to train it is foster：The thick bacterium solution of pod membrane is turned into the liquid that embodiment 2 stops by 10% inoculum concentration
Culture medium, four generation amplification cultivations are carried out, cultivation temperature is 36 DEG C, CO2Throughput is 80%, incubation time 3h, bacterium during harvest
The obvious muddy, OD600 0.53 of liquid；
Step 6, strain harvest, preservation：Bacterium solution is centrifuged, bacterial sediment is collected and is stored in 20% glycerine, dispense, be placed in
Preserved in liquid nitrogen.
Embodiment 9
The invention provides a kind of method for screening streptococcus pneumonia, concretely comprise the following steps：
Step 1, strain is opened in liquid, generation culture：Breakdown streptococcus pneumonia working seed lots strain, with embodiment 3
After the fluid nutrient medium dissolving fungus block of body, fluid present invention culture medium 8ml, 37 DEG C, CO are inoculated in2Throughput is 100%, quiet
Culture 5h is put, this is generation strain；
Step 2, solid line separation, two be commissioned to train it is foster：The solid medium provided in embodiment 6 that generation bacterium solution is transferred is put down
In ware, it is inoculated with line partition method, 37 DEG C, CO2Throughput is 80%, quiescent culture 36h, and this is second-generation bacterial kind；
Step 3, liquid, three generations's culture are passed：The single bacterium colony grown on picking solid medium, it is inoculated in embodiment 3 and carries
In the 3ml Liquid Cultures of confession；With method picking single bacterium colony, 18 are inoculated with fluid nutrient medium respectively, numbering, culture, and cultivation temperature is
37℃、CO2Throughput is 70%, incubation time 8h, cultivate to bacterium solution it is substantially muddy when stop culture, this is three generations's strain；
Step 4, capsule stain, Gram's staining：Each bacterium solution after numbering culture is taken, with India ink staining (gentian violet solution)
Microscopy after dyeing, the bacterium solution of the thick strain reference numeral of pod membrane is retained 3 groups, the bacterium solution of the thin strain reference numeral of remaining pod membrane
It is discarded；
Step 5, pass liquid amplification, four be commissioned to train it is foster：The thick bacterium solution of pod membrane is turned to the liquid of the body of embodiment 3 by 10% inoculum concentration
Body culture medium, four generation amplification cultivations are carried out, cultivation temperature is 35 DEG C, CO2Throughput is 60%, incubation time 4.5h, harvest
When bacterium solution is obvious muddy, OD600 0.60；
Step 6, strain harvest, preservation：Bacterium solution is centrifuged, bacterial sediment is collected and is stored in 15% glycerine, dispense, be placed in
Preserved in liquid nitrogen.
Embodiment 10
Performance test：
(1) strain：The type of S. pneumoniae serotypes 1, the respectively strain before screening and after screening, production strain.
(2) cultivate：Method based on embodiment 8 is opened with fluid nutrient medium, expands three generations, and four generations propagated ferments in 50L
Tank, same medium 20L fermentations, 36 DEG C of cultures, CO2Throughput is 60%, 100rpm stirrings, controls pH7.0, is harvested after 5h.
Bacterium OD values are determined, and do capsule stain, microscopy.Purified after bacterium solution harvest, collect product polysaccharide, and detect European Pharmacopoeia items
Mesh.
(3) experimental result：OD values and polyoses content, qualified situation are as shown in table 3；Thalline capsule stain after culture,
Observed under microscope oil mirror (1000 ×), as shown in Fig. 1~2：
The OD values of table 3 and polyoses content, qualified situation
From table 3 and Fig. 1~2, the strain screened using the present invention is cultivated, and zymotic fluid bacteria concentration is significantly higher than sieve
Strain before choosing, capsular polysaccharide yield also significantly improves after purification, and all meets European Pharmacopoeia indices.
Above-described embodiment, the purpose of the present invention, technical scheme and beneficial effect are carried out further
Describe in detail, should be understood that the embodiment that the foregoing is only the present invention, be not intended to limit the present invention
Protection domain, within the spirit and principles of the invention, any modification, equivalent substitution and improvements done etc., all should include
Within protection scope of the present invention.
Claims (8)
1. the bacterial screening method for pneumonia capsular polysaccharide large-scale production, it is characterised in that comprise the following steps：
Step 1, fluid nutrient medium is prepared：Following raw material is weighed, above-mentioned raw materials are dissolved successively using water for injection, will be adopted
Mixed solution after being dissolved with water for injection carries out constant volume volume needed for, and adjusts pH value, and fluid nutrient medium is obtained after degerming：
Solvent is that the relative usage of water for injection is 1000ml：
It is containing nitrogen source：Trypticase：15~80g, tryptone：20~70g, selected soya peptone：10~80g, soya peptone：10~80g, acid
Caseinhydrolysate：10~70g, yeast extract：15~40g, yeast extract：Any three kinds of compositions in 5~40g；Carbon source
For：Glucose：5.0~50g；Salt is：K2HPO4：0~12.0g, KCl：0~7.0g, Na2HCO3：0~5.0g, NaCl：3.0
~10.0g, MgSO4·7H2O：0.4~5.0g, CaCl2：0~0.14g；Trace element is：L-cysteine hydrochloride：0.12
~1.80g, nicotinic acid：0.5~1.2mg, adenine：5.5~10.5mg, FeSO4：2.5~6.5mg；
Step 2, solid medium is prepared：10~15g of agar is weighed, is dissolved in water for injection rear constant volume volume needed for
90%, the solution after dissolving carries out moist heat sterilization, standby after cooling；
Following raw material is weighed, is dissolved using water for injection, the mixed solution after dissolving is subjected to constant volume volume needed for, and
PH value, bacteria removing are adjusted, is eventually adding in agar medium, pours into plate, solid culture based solvent is made；For the relative of water
Dosage is 1000ml：
It is trypticase containing nitrogen source：15~80g, tryptone：20~70g, selected soya peptone：10~80g, soya peptone：10~80g, acid
Caseinhydrolysate：10~70g, yeast extract：15~40g, yeast extract：Any three kinds in 5~40g；Carbon source is glucose：
5.0~50g；Salt is K2HPO4：0~12.0g, KCl：0~7.0g, Na2HCO3：0~5.0g, NaCl：3.0~10.0g,
MgSO4·7H2O：0.4~5.0g, CaCl2：0~0.14g；Trace element is L-cysteine hydrochloride：0.12~1.80g, cigarette
Acid：0.5~1.2mg, adenine：5.5~10.5mg, FeSO4：2.5~6.5mg；
Step 3, generation culture：Breakdown streptococcus pneumonia working seed lots strain, the fluid nutrient medium prepared with the step 1 are molten
After solving fungus block, fluid nutrient medium 2~20ml, 35 DEG C~37 DEG C, CO prepared by inoculation step 12Throughput is 60%~100%,
4~10h of quiescent culture, obtain generation strain；
Step 4, two be commissioned to train it is foster：Generation bacterium solution is transferred in solid medium plate prepared by step 2, connect with line partition method
Kind, 35 DEG C~37 DEG C, CO2Throughput is 60%~100%, 36~48h of quiescent culture, obtains second-generation bacterial kind；
Step 5, three generations cultivates：The single bacterium colony grown on picking solid medium 15~20, step 1 is inoculated in respectively and is prepared
2~5ml of Liquid Culture in, numbering, culture, cultivation temperature is 35 DEG C~37 DEG C, CO2Throughput is 60%~100%, culture
Time is 8~17h, cultivate to bacterium solution it is substantially muddy when stop culture, obtain three generations's strain；
Step 6, capsule stain, Gram's staining：Each numbering is taken to correspond to bacterium solution after culture, with India ink staining, gentian violet or sand
Microscopy after the dyeing of xanthochromia liquid, the bacterium solution of the thick strain reference numeral of pod membrane is retained 1~5 group, the thin strain of remaining pod membrane is corresponding
The bacterium solution of numbering is discarded；Repeat step 3, step 4 and step 5, again separation screening, the then pure culture filtered out；
Step 7, four be commissioned to train it is foster：The thick bacterium solution of pod membrane is gone to step to the fluid nutrient medium of 1 preparation by 10% inoculum concentration, carried out for four generations
Amplification cultivation, cultivation temperature are 35 DEG C~37 DEG C, CO2Throughput is 60%~100%, and incubation time is 2~5h, treats that bacterium solution is bright
Harvested during aobvious muddy, OD600 >=0.5；
Step 8, strain harvest, preservation：Bacterium solution is centrifuged, bacterial sediment is collected and is stored in 10%~30% glycerine, dispense, put
Preserved in liquid nitrogen.
2. the bacterial screening method according to claim 1 for pneumonia capsular polysaccharide large-scale production, it is characterised in that
In the step 1, the raw material is added in same container, then to Sheng by being stirred continuously in the container of raw mixture
Under the conditions of add water for injection；After water for injection has been added, dissolved by heating, dissolving by heating step is：First with 1 DEG C/min
Heating rate be warming up to 30 DEG C by 25 DEG C, 10min is kept under 30 DEG C of temperature conditionss, then with 1 DEG C/min heating rate
35 DEG C are warming up to by 38 DEG C, 5min is kept under 38 DEG C of temperature conditionss.
3. the bacterial screening method according to claim 1 for pneumonia capsular polysaccharide large-scale production, it is characterised in that
In the step 1 and step 2, mass concentration is used to adjust pH value to 7.0~7.8 for 10% NaOH solution.
4. the bacterial screening method according to claim 1 for pneumonia capsular polysaccharide large-scale production, it is characterised in that
In the step 1, it is filtered under diminished pressure under aseptic technique through 0.1 μm of filtering degerming.
5. the bacterial screening method according to claim 1 for pneumonia capsular polysaccharide large-scale production, it is characterised in that
In the step 2, water for injection is added into agar and is warming up to 95 DEG C and is dissolved；It is in the step 2 that agar solution is cold
But it is standby to 55~60 DEG C.
6. the bacterial screening method according to claim 1 for pneumonia capsular polysaccharide large-scale production, it is characterised in that
In the step 2, moist heat sterilization concrete operations are：Moist heat sterilization temperature is 117~122 DEG C, and pressure is 0.09~0.12MPa,
Sterilization time is 15~30min.
7. the bacterial screening method according to claim 1 for pneumonia capsular polysaccharide large-scale production, it is characterised in that
In the step 2, the bacteria removing with water for injection solution principle is：Depressurized under aseptic technique through 0.2 μm of filtering
Filtration sterilization.
8. the bacterial screening method according to claim 1 for pneumonia capsular polysaccharide large-scale production, it is characterised in that
The strain be used in streptococcus pneumonia 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F,
18C, 19A, 19F, 20,22F, 23F totally 24 serotype.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410007A (en) * | 2001-09-28 | 2003-04-16 | 陈廷伟 | Mannan polysaccharide disease resistant health care beverage |
| WO2008102173A1 (en) * | 2007-02-21 | 2008-08-28 | Health Protection Agency | Compositions comprising polysaccharide conjugates and their use as vaccines |
| CN102093963A (en) * | 2010-11-26 | 2011-06-15 | 兰州生物制品研究所 | Method for culturing streptococcus penumoniae rich in teichoic acid |
-
2017
- 2017-11-30 CN CN201711235794.XA patent/CN107723268A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410007A (en) * | 2001-09-28 | 2003-04-16 | 陈廷伟 | Mannan polysaccharide disease resistant health care beverage |
| WO2008102173A1 (en) * | 2007-02-21 | 2008-08-28 | Health Protection Agency | Compositions comprising polysaccharide conjugates and their use as vaccines |
| CN102093963A (en) * | 2010-11-26 | 2011-06-15 | 兰州生物制品研究所 | Method for culturing streptococcus penumoniae rich in teichoic acid |
Non-Patent Citations (3)
| Title |
|---|
| 张新庄 等: "肺炎链球菌发酵过程中质控方法的应用", 《微生物学免疫学进展》 * |
| 陈天寿: "《微生物培养基的制造与应用》", 30 June 1995 * |
| 黄放 等: "菌种筛选对1型肺炎链球菌产糖量的影响", 《国际生物制品学杂志》 * |
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