CN115466701A - Meningococcus fermentation culture method, fermentation liquid, capsular polysaccharide and application - Google Patents
Meningococcus fermentation culture method, fermentation liquid, capsular polysaccharide and application Download PDFInfo
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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
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Abstract
The application relates to the field of bioengineering, in particular to a meningococcus fermentation culture method, fermentation liquor, capsular polysaccharide and application. The fermentation culture method comprises inoculating meningococcus liquid culture medium with meningococcus liquid culture medium, and performing fermentation culture according to OD of fermentation liquid 600 Value, determining fermentation harvest end point, OD 600 Values are obtained in combination with the capsular polysaccharide yield curve, the bacterial endotoxin curve, and the effective capsular polysaccharide yield curve. The present application has the effect of reducing the endotoxin content of the fermented capsular polysaccharide of meningococcusAnd the effect of increasing capsular polysaccharide production.
Description
Technical Field
The application relates to the field of bioengineering, in particular to a meningococcus fermentation culture method, fermentation liquid, capsular polysaccharide and application.
Background
The scientific name meningococcus (meningococcus) is neisseria meningitidis (n.meninyitidis), a causative bacterium of epidemic cerebrospinal meningitis (epidemic encephalitis). At present, the most effective method for preventing epidemic cerebrospinal meningitis is to inoculate a vaccine, culture meningococcus by fermentation, extract polysaccharide to prepare polysaccharide vaccine, and further achieve the effect of preventing meningitis.
For example, patent document No. 2015105434251 discloses a method for culturing group a meningococcus, in which the strain is cultured and amplified in a semi-complex culture medium, and then the seeds are directly cultured in a fermentation tank of the semi-complex culture medium, but fermentation is directly performed by using the fermentation tank of the semi-complex culture medium, so that the cost of the semi-complex culture medium is high, and the meningococcus after fermentation is low, and thus the method is not suitable for the current requirement for culturing meningococcus.
In view of the above, the prior patent application No. 2021103499492 discloses a culture medium for meningococcus, a preparation method thereof and a culture method, and particularly, by optimizing the formula of the culture medium, acid hydrolyzed casein is reduced on the premise of ensuring the yield and quality of capsular polysaccharide, thereby saving the production cost.
Specifically, the method for culturing the meningococcus comprises the following steps:
s1, preparing a culture medium for the meningitis cocci according to a set formula;
s2, scraping a first meningococcal solid generation of the group A by using a strain rod for solid passage, inoculating the first meningococcal solid generation to a 10% sheep blood agar culture medium plane, uniformly smearing, putting the inoculated 10% sheep blood agar culture medium into an incubator at 37 ℃, and culturing for 12 hours, wherein the culture medium is 7.5% CO2;
s3, sucking the culture medium prepared in the S1 by using a pipette for shake flask culture, and rinsing the lawn on the 10% sheep blood agar culture medium; inoculating the strain in the culture medium prepared in the S1; placing the inoculated shake flask into a large-capacity oscillator, culturing for 6 hours at 37 ℃ and 200rpm until the strain grows into a stationary phase, and finishing the culture.
In view of the above-mentioned related technologies, the inventors believe that during fermentation culture, meningococcus is in the same culture condition in different growth phases, and usually the strain fermentation enters a stationary phase as a fermentation harvest end point, and at this time, the strain grows excessively, so that the strain dies excessively during fermentation, and the content of bacterial endotoxin generated by growth and metabolism of meningococcus is high.
Bacterial vaccine capsular polysaccharides are difficult to remove bacterial endotoxin in the later process. The prior art removes the bacterial endotoxin below the quality standard, and a complex series of processes are required. On one hand, organic reagents or other foreign substances are added in the processes, and if the impurity removal rate is not completely removed, new vaccine side reactions can be caused; on the other hand, the increase of the process for removing the bacterial endotoxin has the influence on the reduction of the yield of the capsular polysaccharide. Therefore, how to avoid the generation of bacterial endotoxin and control the generation of bacterial endotoxin under a lower quality standard so as to further reduce the adverse reaction rate of vaccine finished products such as fever, red swelling, nodules and the like, and is a permanent technical subject of bacterial vaccines.
Disclosure of Invention
In order to reduce the endotoxin content of the capsular polysaccharide of meningococcus fermentation, the application provides a meningococcus fermentation culture method.
In a first aspect, the application provides a meningococcal fermentation culture method, which adopts the following technical scheme:
a method for culturing meningococcus by fermentation comprises inoculating meningococcus liquid culture liquid to meningococcus liquid culture medium, and culturing by fermentation according to OD of the fermentation liquid 600 And (4) judging the fermentation harvest end point.
By adopting the scheme: according to OD of fermentation broth 600 And (4) judging the fermentation harvest end point, so that the high-quality capsular polysaccharide can be accurately controlled to be harvested, on one hand, the yield of the capsular polysaccharide can be improved, and on the other hand, the bacterial endotoxin generated by metabolism due to excessive death of bacteria in the subsequent fermentation process can be reduced.
In addition, the whole culture fermentation process is controllable by directly fermenting and culturing liquid culture bacteria, and the quality and the yield of the obtained capsular polysaccharide product can be improved.
Optionally, OD 600 When the OD reached 5.0 or more, the OD was monitored 600 Value, OD 600 When the value reaches 5.0-6.0, the meningococcal fermentation broth is harvested.
By adopting the scheme, when OD is performed 600 The meningococcal fermentation liquor obtained when the value reaches 5.0-6.0 has less dead bacteria, so that the content of endotoxin in subsequently obtained capsular polysaccharide is greatly reduced, and the quality is higher.
Alternatively, OD 600 When 5.0 was reached, OD was monitored 600 Value, OD 600 When the value reaches 5.5-6.0, the meningococcus A group, C group and W135 group fermentation liquid is harvested.
By adopting the scheme, the OD is specifically designed for fermentation harvest of meningococcus A group, meningococcus C group and meningococcus W135 group fermentation liquid 600 The timing of harvest is more suitable, so that meningococcal group a, group C and group W135 capsular polysaccharides with less endotoxin are obtained.
Alternatively, OD 600 When 5.0 was reached, OD was monitored 600 Value, OD 600 At values of 5.5-5.7, meningococcal groups a and C were harvested.
By adopting the scheme, the fermentation harvest OD designed for meningococcus A group and C group fermentation liquid is specifically designed 600 The harvesting time is more suitable, so that meningococcal group A and C capsular polysaccharide with less endotoxin is obtained.
Optionally, OD 600 When 5.0 was reached, OD was monitored 600 Value, OD 600 When the value reaches 5.8-6.0, the meningococcal W135 fermentation liquid is harvested.
By adopting the scheme, the OD is obtained by targeted fermentation designed for meningococcus W135 group fermentation liquid 600 The harvesting time is more suitable, so that the meningococcal W135 group capsular polysaccharide with less endotoxin is obtained.
Optionally, OD 600 When the OD reached 5.0 or more, the OD was monitored 600 Value, OD 600 When the value reaches 5.0-5.5, the meningococcal Y fermentation liquid is harvested.
By adopting the scheme, the OD is obtained by specifically designing the meningococcus Y-group fermentation liquid 600 The harvesting time is more suitable, so that the meningococcal group Y capsular polysaccharide with less endotoxin is obtained.
Optionally, the meningococcus liquid culture medium is inoculated with the meningococcus liquid culture medium, and fermentation culture is carried out at the temperature of 37 +/-1.5 ℃, the pH value of 6.8-7.4 and the rotation speed of 100-300 rpm.
In a second aspect, the present application provides a meningococcal fermentation broth prepared by the above fermentation culture method.
In a third aspect, the application provides a refined polysaccharide prepared from a meningococcal fermentation broth.
In a fourth aspect, the 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. according to the method, the judgment of the fermentation harvest end point is controlled, the high-quality capsular polysaccharide can be accurately harvested, and bacterial endotoxin generated by metabolism due to excessive death of bacteria in the subsequent fermentation process is reduced.
2. The application is based on different OD in the culture process 600 The culture conditions are adjusted, so that a proper growth environment is provided for the strains in each culture stage, the death of the strains in the fermentation process is reduced, the production of bacterial endotoxin is reduced, and the yield is improved.
Drawings
FIG. 1 is a graph of polysaccharide content and endotoxin content of a meningococcus group A of the present application;
FIG. 2 is a graph of polysaccharide content and endotoxin content of group C meningococci of the present application;
FIG. 3 is a graph of polysaccharide content and endotoxin content of meningococcus group Y of the present application;
FIG. 4 is a graph of polysaccharide content and endotoxin content of meningococcus group W135 of the present application.
Detailed Description
The applicant finds that a certain delicate relationship exists between a curve generated by bacterial endotoxin along with growth metabolism and a curve generated by capsular polysaccharide through research and exploration. The former industry technicians only pursue the yield or only pay attention to the yield curve of the capsular polysaccharide content; or the harvest end point is controlled at the beginning of the stationary phase by focusing on the rapid increase of bacterial endotoxin in the stationary phase. Applicants have discovered a point of equilibrium between the bacterial endotoxin profile and the capsular polysaccharide production profile by aggregating, through constant experimental exploration, the metabolic profiles of the different meningococcal serotypes, by controlling the OD of the fermentation harvest point 600 In this regionIn addition, the method can realize the control of the bacterial endotoxin at a lower level on the premise of not reducing the yield and the quality of the capsular polysaccharide.
Meanwhile, the applicant also finds that a curve of the yield of the meningococcal capsular polysaccharide has a light and dark curve, and the curve on the bright surface (called a bright line for short) refers to the total content of the capsular polysaccharide in the fermentation liquid detected on the surface. The total content of the capsular polysaccharide of the open line, including the broken chains of capsular polysaccharide fragments and the polysaccharide yield after metabolic decomposition, the polysaccharide chains are often short and small fragments and are non-effective components or low-effective components of vaccine antigen components, actually, the effective components of the capsular polysaccharide are large molecular fragments (generally, the molecular weight is required to be more than 200 KD), the curve presented during fermentation growth is often different from the open line, and the invention is called as the dark line. Thus, the fermentation end-point OD of the present application 600 Values are obtained in combination with the capsular polysaccharide yield curve, the bacterial endotoxin curve, and the effective capsular polysaccharide yield curve.
The present application will be described in further detail with reference to the following drawings and examples.
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 strain A4CMCC29201, neisseria meningitidis strain C11CMCC29205, neisseria meningitidis strain Y YCMCC29303, neisseria meningitidis strain W135CMCC29055.
Preparation of culture Medium for meningococcus
Preparation example 1
The culture medium for the meningitis cocci is prepared from a liquid A and a liquid B according to a volume ratio of 9. 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, the first and the second end of the pipe are connected with each other,
each liter of the A liquid 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 of sodium L-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 meningitis cocci.
It should be noted that the content and ratio of the raw material formula can be adjusted adaptively according to the actual situation, for example, the culture medium of meningococcus can be, but 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 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 meningococcus comprises the following steps:
(1) Weighing acid hydrolyzed casein, yeast leaching powder, L-cystine, monopotassium phosphate, sodium dihydrogen phosphate, glycine, L-sodium glutamate, ammonium chloride and sodium chloride according to the preparation amount, adding water for injection into a container for dissolving, and then adjusting the pH value to 7.3 by using 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 sterilizing cabinet at 115 deg.C for 30min to obtain culture medium for meningococcus.
Examples
The meningococcal fermentation culture method provided in embodiment 1 includes the following steps:
s1, liquid recovery culture
Starting group A, group C, group Y and group W135 strains respectively, inoculating the group A, the group C, the group Y and the group W135 strains to the meningococcus culture medium provided by the preparation example 1 respectively, and inoculating the group A, the group C, the group Y and the group W to the meningococcus culture medium provided by the preparation example 1, wherein the culture temperature is 37 +/-1.5 ℃, the culture process is carried out at the culture pH of 6.5, the rotating speed is 200rpm for 3 hours, and the OD is 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 broth into the culture medium for meningococcus provided in preparation example 1, culturing at 37 + -1.5 deg.C under controlled pH of 6.5, culturing at 200rpm for 3 hr, and culturing at OD 600 When the culture reaches 1.0, finishing the culture to obtain the second-generation liquid culture bacterial liquid.
S3, fermentation culture
The second-generation liquid culture broth was inoculated into 10-fold volume of the culture medium for meningococcus provided in preparation example 1 for fermentation culture, and OD was monitored 600 Value, OD 600 At a value of 5.0, groups a, C, Y and W135 meningococcal fermentation broths were harvested.
The application also provides refined polysaccharide prepared by meningococcal fermentation broth, taking the preparation of A group meningococcal refined polysaccharide by A group meningococcal fermentation broth as an example, the method comprises the following steps:
a formaldehyde solution was added to the group A meningococcal fermentation broth harvested in example 1 at 1% (v/v), sterilized for 60 minutes, centrifuged at 4000rpm in a centrifuge at 10 ℃ for 15min to remove the bacteria, and the supernatant was collected. 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 for 15 minutes each, centrifuged at 15 ℃ or below) in a volume of 2 times that of the polysaccharide, and washed twice with 2-8 ℃ pre-cooled acetone (4000rpm for 15 minutes each, centrifuged at 15 ℃ or below) in a volume of 2 times that of the polysaccharide. And (4) discarding the supernatant, collecting polysaccharide precipitate, and drying in vacuum or by blow-drying to obtain the crude product of the meningococcal group A capsular polysaccharide.
Dissolving the crude product of the meningococcus group A capsular polysaccharide in 10% saturated neutral sodium acetate solution to make the concentration of the crude product of the meningococcus group A capsular polysaccharide reach 10-15 mg/ml, and extracting for 3-12 times by using 10% saturated neutral sodium acetate cold phenol solution which is 2 times of the volume of the crude product of the meningococcus group A capsular polysaccharide. 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 ultrafiltering and dialyzing with 10-20 times volume of injection water (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 ℃ to a final concentration of: 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 A-group meningococcal refined polysaccharide.
The application also provides a meningococcal polysaccharide vaccine or conjugate vaccine prepared from the meningococcal refined polysaccharide. In particular, the preparation method of the vaccine is a method and a process commonly used by those in the art, and is not described herein.
In addition, it should be noted that the focus of the present application is on the decision option of fermentation harvest end point, and the culture medium for meningococcus provided by the present application is only a supplement to the integrity of the technical solution.
Example 2
Example 2 differs from example 1 in that in step S3, the OD is monitored 600 Value, OD 600 At a value of 5.2, meningococcal group a, group C, group Y and group W135 broths were harvested.
Example 3
Example 3 differs from example 1 in that in step S3, the OD is monitored 600 Value, OD 600 At a value of 5.5, fermentation broths from meningococcal groups a, C, Y and W135 were harvested.
Example 4
Example 4 differs from example 1 in that in step S3, the OD is monitored 600 Value, OD 600 At a value of 5.7, fermentation broths from meningococcal groups a, C, Y and W135 were harvested.
Example 5
Example 4 differs from example 1 in that in step S3, the OD is monitored 600 Value, OD 600 At a value of 5.8, fermentation broths from meningococcal groups a, C, Y and W135 were harvested.
Example 6
Example 6 differs from example 1 in that in step S3, the OD is monitored 600 Value, OD 600 At a value of 6.0, meningococcal groups A, C, Y andfermentation broth of W135 population.
Example 7
Example 7 differs from example 1 in that in step S3, the OD is monitored 600 Value, OD 600 At a value of 6.2, fermentation broths from meningococcal groups a, C, Y and W135 were harvested.
Example 8
Example 8 differs from example 1 in that in step S3, the OD is monitored 600 Value, OD 600 At a value of 4.8, fermentation broths from meningococcal groups a, C, Y and W135 were harvested.
Example 9
Example 9 differs from example 1 in that in step S3, OD is used 600 When the temperature reaches above 3.0, fermentation culture is carried out by controlling the temperature to be 33 +/-1.5 ℃, the pH value to be 6.8, the rotating speed to be 300rpm and the dissolved oxygen to be 20 percent.
Example 10
Example 10 differs from example 9 in that in step S3, OD is used 600 When the temperature reaches above 3.0, fermentation culture is carried out by controlling the temperature to be 33 +/-1.5 ℃, the pH value to be 7.0, the rotating speed to be 300rpm and the dissolved oxygen to be 20 percent.
Example 11
Example 11 differs from example 9 in that in step S3, OD is used 600 When the temperature reaches above 3.0, the fermentation culture is carried out by controlling the temperature to be 33 +/-1.5 ℃, the pH value to be 7.2, the rotating speed to be 300rpm and the dissolved oxygen to be 20 percent.
Example 12
Example 12 differs from example 9 in that in step S3, OD is used 600 When the temperature reaches above 3.0, the fermentation culture is carried out by controlling the temperature to be 33 +/-1.5 ℃, the pH value to be 6.8, the rotating speed to be 100rpm and the dissolved oxygen to be 20 percent.
Example 13
Example 13 differs from example 9 in that in step S3, OD is used 600 When the temperature reaches above 3.0, fermentation culture is carried out by controlling the temperature to be 33 +/-1.5 ℃, the pH value to be 6.8, the rotating speed to be 300rpm and the dissolved oxygen to be 30 percent.
Example 14
Example 14 differs from example 11 in that in step S3, OD is used 600 When the temperature reaches above 3.0, the temperature is controlled to be 33 +/-1.5 ℃, the pH value is controlled to be 6.8, the rotating speed is 300rpm, and the dissolved oxygen is 40 percentAnd performing fermentation culture.
Example 15
Example 15 differs from example 1 in that in step S3, OD is used 600 When the temperature reaches above 4.0, the fermentation culture is carried out by controlling the temperature to be 31 +/-1.5 ℃, the pH value to be 6.8, the rotating speed to be 300rpm and the dissolved oxygen to be 40 percent.
Example 16
Example 16 differs from example 1 in that in step S3, OD is used 600 When the temperature reaches above 4.0, fermentation culture is carried out by controlling the temperature to be 31 +/-1.5 ℃, the pH value to be 7.0, the rotating speed to be 300rpm and the dissolved oxygen to be 40 percent.
Example 17
Example 9 differs from example 16 in that in step S3, OD is used 600 When the temperature reaches above 4.0, the fermentation culture is carried out by controlling the temperature to be 31 +/-1.5 ℃, the pH value to be 7.2, the rotating speed to be 300rpm and the dissolved oxygen to be 40 percent.
Example 18
Example 18 differs from example 16 in that in step S3, OD is used 600 When the temperature reaches above 4.0, controlling the temperature to be 31 +/-1.5 ℃, the pH value to be 6.8, rotating speed to be 100rpm and dissolved oxygen to be 40 percent for fermentation culture;
example 19
Example 19 differs from example 18 in that, in step S3, OD is measured 600 When the temperature reaches above 4.0, controlling the temperature to be 31 +/-1.5 ℃, the pH value to be 7.0, rotating speed to be 100rpm and dissolved oxygen to be 50 percent for fermentation culture;
example 20
Example 20 differs from example 1 in that in step S3, OD is measured 600 When the temperature reaches above 3.0, controlling the temperature to be 33 +/-1.5 ℃, the pH value to be 6.8, rotating speed to be 300rpm and dissolved oxygen to be 40 percent for fermentation culture; when OD is reached 600 When the temperature reaches above 4.0, fermentation culture is carried out by controlling the temperature to be 31 +/-1.5 ℃, the pH value to be 7.0, the rotating speed to be 100rpm and the dissolved oxygen to be 40 percent.
Example 21
Example 21 differs from example 1 in that, in step S3, OD is measured 600 When the temperature reaches above 3.0, controlling the temperature to be 33 +/-1.5 ℃, controlling the pH to be 6.8, rotating speed to be 300rpm, and carrying out fermentation culture on 30% of dissolved oxygen; when OD is reached 600 When the temperature reaches above 4.0, the fermentation culture is carried out by controlling the temperature to be 31 +/-1.5 ℃, the pH value to be 7.0, the rotating speed to be 100rpm and the dissolved oxygen to be 40 percent.
Example 22
Example 22 differs from example 1 in that in step S3, OD is used 600 When the temperature reaches above 3.0, controlling the temperature to be 33 +/-1.5 ℃, the pH value to be 6.8, rotating speed to be 300rpm, and carrying out fermentation culture by dissolved oxygen of 20 percent; when OD is reached 600 When the temperature reaches above 4.0, fermentation culture is carried out by controlling the temperature to be 31 +/-1.5 ℃, the pH value to be 6.8, the rotating speed to be 100rpm and the dissolved oxygen to be 40 percent.
Example 23
Example 23 differs from example 1 in that, in step S3, OD is measured 600 When the temperature reaches above 3.0, controlling the temperature to be 33 +/-1.5 ℃, the pH value to be 6.8, rotating speed to be 300rpm and dissolved oxygen to be 40 percent for fermentation culture; when OD is reached 600 When the temperature reaches above 4.0, fermentation culture is carried out by controlling the temperature to be 31 +/-1.5 ℃, the pH value to be 6.8, the rotating speed to be 100rpm and the dissolved oxygen to be 40 percent.
Example 24
Example 24 differs from example 23 in that the culture medium for meningococcus provided in preparation example 2 was used in steps S1, S2 and S3.
Comparative example
Comparative example 1
The meningococcus fermentation culture method provided by the comparative example 1 comprises the following steps:
s1, liquid recovery culture
Starting group A, group C, group Y and group W135 strains respectively, inoculating the group A, the group C, the group Y and the group W135 strains to the meningococcus culture medium provided by the preparation example 1 respectively, inoculating the group A, the group C, the group Y and the group W to the meningococcus culture medium provided by the preparation example 1, culturing at the temperature of 37 +/-1.5 ℃, controlling the culture pH to be 6.5 by glacial acetic acid in the culturing process, culturing for 3 hours at the rotating speed of 200rpm, and culturing the strains at 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 6.5 with glacial acetic acid, and cultured at 200rpm for 3 hours at OD 600 When the culture 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 the meningococcal culture medium provided in preparation example 1 for fermentation culture, controlling dissolved oxygen to be 20% until the bacterial strain grows into a stable phase, finishing the culture, and harvesting group A, C, Y and W135 meningococcal fermentation liquid.
Comparative example 2
The difference from the embodiment 1 is that,
s3, fermentation culture
Inoculating the second generation liquid culture broth into the culture medium of meningococcus provided in preparation example 1, and performing fermentation culture when OD is obtained 600 When the temperature reaches above 3.0, controlling the temperature to be 33 plus or minus 1.5 ℃, the pH value to be 6.8, rotating speed to be 300rpm, and carrying out fermentation culture by dissolved oxygen of 40 percent until the strain grows into a stable period, and finishing the culture.
Comparative example 3
The difference from the embodiment 1 is that,
s3, fermentation culture
Inoculating the second generation liquid culture broth into the culture medium of meningococcus provided in preparation example 1, and performing fermentation culture when OD is obtained 600 When the temperature reaches above 4.0, controlling the temperature to be 31 plus or minus 1.5 ℃, the pH value to be 6.8, rotating speed to be 100rpm, and carrying out fermentation culture by dissolved oxygen of 40 percent until the strain grows into a stable period, and finishing the culture.
Comparative example 4
The difference from the embodiment 1 is that,
s3, fermentation culture
Inoculating the second generation liquid culture broth into the culture medium of meningococcus provided in preparation example 1, and performing fermentation culture when OD is obtained 600 When the temperature reaches above 3.0, controlling the temperature to be 33 +/-1.5 ℃, controlling the pH to be 6.8, rotating speed to be 100rpm, and carrying out fermentation culture on dissolved oxygen of 40%; when OD is reached 600 When the temperature reaches above 4.0, controlling the temperature to be 31 plus or minus 1.5 ℃, the pH value to be 6.8, rotating speed to be 300rpm, and carrying out fermentation culture by dissolved oxygen of 40 percent until the strain grows into a stable period, and finishing the culture.
Comparative example 5
The meningococcus fermentation culture method provided by the comparative example 5 comprises the following steps:
s1, solid recovery culture
Scraping A, C, Y and W135 group meningococcal solid generation with strain rod, respectively, and inoculating to 10% sheepUniformly spreading on the surface of blood agar culture medium, placing the inoculated 10% sheep blood agar culture medium at 37 deg.C, 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-capacity shaker and incubated at 37 ℃ for 6 hours at 200 rpm.
And S3, inoculating the second-generation liquid culture bacterium liquid into a meningococcal culture medium for fermentation culture, culturing at the temperature of 37 +/-1.5 ℃ and the pH of 6.9 at the rotating speed of 300rpm until the strain grows into a stable period, finishing the culture, and harvesting the A, C, Y and W135 group meningococcal fermentation liquids.
Performance detection
1. Bacterial endotoxin:
when the bacterial endotoxin test is carried out by adopting a method 1143 of general rules of China pharmacopoeia of 2020 edition, polysaccharides of group A, group C, group Y and group W135 are not higher than 12.5EU/ug, and the results are shown in table 1.
2. Effective yield test of refined polysaccharide:
1) Total capsular polysaccharide content the purified polysaccharides of examples 2-23 and comparative examples 1-4 were obtained according to the preparation method of the purified polysaccharide provided in example 1, and the polysaccharides of examples 1-23 and comparative examples 1-5 were weighed, respectively, and the results of the measurements are shown in Table 2.
2) Effective capsular polysaccharide content:
group A with molecular weight greater than or equal to 200KD
Group C with molecular weight greater than or equal to 200KD
Group Y with molecular weight of 245KD or more
W135 group with molecular weight of 270KD or more
TABLE 1 bacterial endotoxin assay Table
TABLE 2 refined 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: inoculating the liquid culture solution of meningococcus to the liquid culture medium of meningococcus, fermenting and culturing according to OD of the fermentation liquid 600 And (4) judging the fermentation harvest end point.
2. The meningococcal fermentation culture process of claim 1, wherein: OD 600 When the OD reached 5.0 or more, the OD was monitored 600 Value, OD 600 When the value reaches 5.0-6.0, the meningococcal fermentation broth is harvested.
3. The meningococcal fermentation culture process of claim 2, wherein: OD 600 When 5.0 was reached, OD was monitored 600 Value, OD 600 At values of 5.5-6.0, meningococcal group a, group C and group W135 fermentations were harvested.
4. The meningococcal fermentation culture process of claim 3, wherein: OD 600 When 5.0 was reached, OD was monitored 600 Value, OD 600 When the value reaches 5.5-5.7, the meningococcus A group and C group fermentation liquid is harvested.
5. According to the claimsThe meningococcal fermentation culture method of claim 3, characterized by: OD 600 When 5.0 was reached, OD was monitored 600 Value, OD 600 When the value reaches 5.8-6.0, the meningococcal W135 fermentation liquid is harvested.
6. The meningococcal fermentation culture process of claim 2, wherein: OD 600 When the OD reached 5.0 or more, the OD was monitored 600 Value, OD 600 When the value reaches 5.0-5.5, the meningococcal Y fermentation liquid is harvested.
7. The meningococcal fermentation culture process of claim 1, wherein: inoculating the liquid culture solution of the meningococcus to a culture medium of the meningococcus, and performing fermentation culture at the temperature of 37 +/-1.5 ℃, the pH value of 6.8-7.4 and the rotating speed of 100-300 rpm.
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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