CN117821342A - Fermentation method of 14 type streptococcus pneumoniae - Google Patents
Fermentation method of 14 type streptococcus pneumoniae Download PDFInfo
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- CN117821342A CN117821342A CN202410226966.0A CN202410226966A CN117821342A CN 117821342 A CN117821342 A CN 117821342A CN 202410226966 A CN202410226966 A CN 202410226966A CN 117821342 A CN117821342 A CN 117821342A
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- 241000193998 Streptococcus pneumoniae Species 0.000 title claims abstract description 136
- 229940031000 streptococcus pneumoniae Drugs 0.000 title claims abstract description 136
- 238000000034 method Methods 0.000 title claims abstract description 56
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention relates to the technical field of fermentation engineering, in particular to a fermentation method of type 14 streptococcus pneumoniae. The invention remarkably improves the capsular polysaccharide yield of the 14 type streptococcus pneumoniae by optimizing the feed supplement culture medium used in the fermentation process of the 14 type streptococcus pneumoniae, and further improves the capsular polysaccharide yield by matching with the fermentation condition optimization. The fermentation method of the 14 type streptococcus pneumoniae provided by the invention can be used for preparing streptococcus pneumoniae capsular polysaccharide vaccines and capsular polysaccharide conjugate vaccines, and has a good application prospect.
Description
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to a fermentation method of type 14 streptococcus pneumoniae.
Background
Streptococcus pneumoniaeStreptococcus pneumoniae) Is a gram positive coccus with capsule, and is the main pathogenic bacteria for human pneumonia, meningitis, tympanitis, bacteremia and other diseases. The diseases caused by streptococcus pneumoniae have higher morbidity and mortality, and the main susceptible population is infants under 5 years old. With the increasing development of Streptococcus pneumoniae resistance, protection against infection by vaccination is increasingly important. The capsular polysaccharide is a key virulence factor of streptococcus pneumoniae, and after the streptococcus pneumoniae is cultured in a fermentation tank, the polysaccharide or polysaccharide combined vaccine prepared by extraction, separation, purification and combination can effectively induce human immune response, thereby preventing infection and morbidity of the streptococcus pneumoniae.
In the production of capsular polysaccharides from pneumonias, fermentation of streptococcus pneumoniae is a key step affecting capsular polysaccharide production. Fermentation of microorganisms is a very complex system, affected by a number of factors, among which the main influencing factors include: strain, medium composition, feed medium composition, culture pH, culture temperature, aeration and agitation, batch, feed or fed-batch, etc. Meanwhile, the synthesis of streptococcus pneumoniae capsular polysaccharide is the result of comprehensive regulation and control by a plurality of factors, and the metabolic pathway is complex. Studies have shown that the yield of capsular polysaccharide from Streptococcus pneumoniae is not in a simple proportional relationship with the concentration of the bacterial cells, i.e., the yield of capsular polysaccharide is not necessarily the highest under the most suitable growth conditions of Streptococcus pneumoniae (Chai Yanjing, levozhijie, ma Bo, etc. optimization of Streptococcus pneumoniae type 5 fermentation process [ J ]. Modern biomedical advances, 2012,12 (09): 1660-1664.DOI: 10.13241/j.cnki.pmb.2012.09.002). Thus, there is a need for co-optimization by multiple influencing factors to achieve an optimal combination of streptococcus pneumoniae growth and capsular polysaccharide synthesis.
There are significant differences in the fermentation processes required for the production of capsular polysaccharides by different types of streptococcus pneumoniae. In the existing fermentation process of streptococcus pneumoniae, the yield of the type 14 streptococcus pneumoniae capsular polysaccharide is still low. Patent application WO2011151841A1 discloses a Streptococcus pneumoniae fermentation process wherein the type 14 Streptococcus pneumoniae can achieve a biomass of 0.9-15.0 and a polysaccharide yield of 0.2-0.25g/L during fermentation; in the studies of Maria et al, the yield of streptococcus pneumoniae polysaccharide 14 can reach 185.2 mg/L (LEAL, m.m.; PEREIRA, d.s.g.; JESSOUROUN, e.; COUTO, m.a. p.g. and PEREIRA, n. (2011) Investigation of cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 14. Electronic Journal of Biotechnology, vol.14, no.5, http:// dx.doi.org/10.2225/vol14-issue 5-fulltext-6) by optimization of the culture medium and the culture process; patent application CN108473945A discloses the growth density OD of Streptococcus pneumoniae 14 by using a modified AS3 medium, controlling the fermentation temperature at 36℃and pH at 7.0, using NaOH AS an alkaline titrant, inerting the fermenter with air, and stirring the fermentation at 200 rpm 600 Can reach 6.8, and the fermentation yield of polysaccharide can reach 1.1g/L.
Studies have shown that perfusion fermentation can circulate the culture through a perfusion system, remove spent media and waste products, and maintain culture volume by introducing fresh media, and greatly increase the growth density, OD, of Streptococcus pneumoniae 600 Can reach 30, and the corresponding polysaccharide yield can reach 2.2 g/L (see patent application CN 108473945A). However, since the operation of the perfusion fermentation is complicated and the consumable cost is high and the aseptic operation during the circulation of the perfusion system outside the fermenter is very complicated, two modes of Batch fermentation (Batch) or Fed-Batch fermentation (Fed-Batch) are often used in the actual production process. Patent application CN104080904A discloses that the number of streptococcus pneumoniae capsules 14 is optimized by a series of processesThe batch fermentation yield of sugar is 450mg/L, and the fed-batch fermentation can reach 1100 mg/L.
In view of the foregoing, there is a need for further optimization of the current streptococcus pneumoniae type 14 fermentation process to increase the yield of capsular polysaccharides.
Disclosure of Invention
The invention provides a fermentation method of type 14 streptococcus pneumoniae.
Specifically, the invention provides the following technical scheme:
the invention provides a fermentation method of streptococcus pneumoniae type 14, which comprises the following steps: in the fermentation process of the streptococcus pneumoniae type 14, feeding is carried out by a feeding culture medium;
the feed medium comprises the following components: 180-220g/L glucose, 100-150g/L yeast extract, 100-150g/L soybean peptone, 1.1-1.5g/L amino acid, 0.3-0.8g/L magnesium salt, 0.05-0.2g/L ferrous salt, 0.01-0.02g/L zinc salt and 5-10g/L manganese salt.
In order to increase the yield of capsular polysaccharide of streptococcus pneumoniae type 14, the invention first optimizes the feed medium. In the research and development process, the invention discovers that, unlike the prior general cognition (the growth and metabolism of the streptococcus pneumoniae in the middle and later stages of fermentation are slow, the required nutrition is reduced, high-concentration carbon and nitrogen source feed is not needed, for example, WO2011151841A 1) is adopted in the feed supplementing process, and the growth of the streptococcus pneumoniae type 14 and the synthesis of capsular polysaccharide can be balanced better by matching with the supply of inorganic salt ions (particularly high-concentration manganese ions) with specific types and concentrations by adopting the higher-concentration carbon source and nitrogen source in the feed supplementing process, so that the yield of capsular polysaccharide is obviously improved. Experiments prove that the yield of capsular polysaccharide is obviously improved by controlling the carbon source, the nitrogen source and the inorganic salt in the feed medium within the concentration range.
The amino acids mentioned above include L-glutamine and asparagine.
Preferably, the feed medium further comprises choline chloride and a reducing agent.
As for the selection of the above-mentioned inorganic salts, it is known to those skilled in the art that the above-mentioned inorganic salts function to provide the corresponding metal ions (magnesium ions, ferrous ions, zinc ions, manganese ions), and thus any salt of the above-mentioned metal ions can achieve the desired effect. Illustratively, the magnesium salt may be selected from one or more of magnesium sulfate, magnesium chloride, magnesium acetate, the ferrous salt may be selected from one or more of ferrous sulfate, ferrous chloride, ferrous acetate, the zinc salt may be selected from one or more of zinc sulfate, zinc chloride, zinc acetate, and the manganese salt may be selected from one or more of manganese sulfate, manganese chloride.
Preferably, the feed medium comprises the following components: 180-220g/L glucose, 100-150g/L yeast extract, 100-150g/L soybean peptone, 0.46-0.52 g/L-glutamine, 0.7-0.9g/L asparagine and MgSO 4 ·7H 2 O 0.45-0.53g/L，FeSO 4 ·7H 2 O 0.09-0.11g/L，ZnSO 4 ·7H 2 O 0.015-0.017g/L，MnSO 4 ·H 2 O6.8-7.6 g/L, thioglycollic acid 0.18-0.22mL/L, choline chloride 0.01-0.03 g/L.
The inorganic salt in the feed medium may be replaced with other salt forms corresponding to the metal ions (for example, sulfate may be replaced with hydrochloride, acetate, etc.), and the concentration of the inorganic salt after replacement may be converted according to the concentration and molecular weight of the metal ions contained in the sulfate. It will be appreciated by those skilled in the art that substitution of different salt species of the same metal ion should be equivalent to the technical solution of the present invention and also fall within the scope of the present invention. The inorganic salt may be a crystalline hydrate of the inorganic salt or an inorganic salt without crystal water, and the amount of the corresponding inorganic salt without crystal water may be converted by those skilled in the art based on the amount of the crystalline hydrate.
In some embodiments of the invention, the feed medium comprises the following components: 200+ -10g/L glucose, 100-150g/L yeast extract, 100-150g/L soybean peptone, 0.496+ -0.02 g/L-glutamine, 0.8+ -0.04 g/L asparagine, 0.02+ -0.001 g/L choline chloride, and MgSO 4 ·7H 2 O 0.5±0.025g/L，FeSO 4 ·7H 2 O 0.1±0.005g/L，ZnSO 4 ·7H 2 O 0.016±0.0008g/L，MnSO 4 ·H 2 O7.2+/-0.36 g/L, thioglycollic acid 0.2+/-0.01 ml/L.
Preferably, the pH of the feed medium is from 6.6 to 7.3. The solvent of the feed medium is water.
In the fermentation method, the feeding is continuous feeding, and the feeding speed is based on the condition that the concentration of glucose in a fermentation system is maintained to be not lower than 5 g/L.
Illustratively, the rate of feed is based on maintaining a concentration of glucose in the fermentation system of 5-10g/L for cost reasons.
Preferably, the fermentation is carried out at a cell density (OD 694 ) When the concentration of glucose in the fermentation system is more than 3 or is lower than 15g/L, the feeding culture medium is used for feeding.
In the present invention, the fermentation process is fed-batch fermentation.
As the fermentation medium used in the above fermentation method, a medium known to those skilled in the art for fermentation of Streptococcus pneumoniae type 14 can be used, for example: a fermentation medium described on page 14 of the specification of patent application WO2011151841A1, or fermentation media described on pages 16 to 17 of the specification of patent application CN108473945A, and the like.
In some embodiments of the invention, the fermentation medium comprises the following components: 10-50g/L glucose, 10-50g/L yeast extract, 10-50g/L soybean peptone, 1-10g/L dipotassium hydrogen phosphate, 0.5-5g/L sodium bicarbonate, 0.01-0.1. 0.1 g/L-glutamine, 0.05-0.2g/L asparagine, 0.005-0.02g/L choline chloride, and MgSO 4 ·7H 2 O 0.01-0.2g/L，FeSO 4 ·7H 2 O 0.01-0.2g/L，ZnSO 4 ·7H 2 O 0.001-0.01g/L，MnSO 4 ·H 2 0.001-0.01g/L O, 0.05-0.2ml/L thioglycollic acid.
Based on the feed medium, the invention further optimizes the fermentation conditions. Streptococcus pneumoniae is a facultative anaerobe, and at present, air or oxygen and a mixture thereof with carbon dioxide or carbon dioxide are commonly used for ventilation in the fermentation process. The invention has been unexpectedly found in the development process that the nitrogen gas is introduced in the fermentation process of the streptococcus pneumoniae type 14 to significantly improve the yield of capsular polysaccharide.
Preferably, in the fermentation method described above, the gas environment of the fermentation is nitrogen.
Further preferably, during the fermentation, nitrogen is introduced at an aeration rate of 0.08 to 0.12 VVM (more preferably 0.1.+ -. 0.005 VVM).
In combination with the optimization of the feed medium and the fermentation aeration, the invention determines the optimal fermentation temperature, pH and rotation speed through screening.
Preferably, the temperature of the fermentation is 36.5-37.5 ℃. More preferably 37.+ -. 0.1 ℃. Compared with other fermentation temperatures, the streptococcus pneumoniae type 14 can more stably produce capsular polysaccharide at the fermentation temperature, and the process robustness is improved.
The pH of the fermentation is 6.6-7.3. More preferably 6.8.+ -. 0.1.
The rotation speed of the fermentation is 80-100rpm. More preferably 90.+ -.5 rpm.
In the above fermentation method, the fermentation is preferably ended at the late stage of fermentation. The late fermentation period refers to the late logarithmic growth period of fermentation, and can be referred to OD of streptococcus pneumoniae fermentation liquid 694 And reaching the stage to judge the fermentation later stage.
In order to obtain fermented inoculation bacterial liquid, the method further comprises the steps of resuscitating bacterial in a solid culture medium and transferring the resuscitated bacterial to a liquid culture medium for amplification culture before the fermentation.
The recovery of the strains in the solid medium can be achieved by technical means known to the person skilled in the art, for example Chai Yanjing et al in the "optimization of Streptococcus pneumoniae type 5 fermentation process" 1.4 culture section disclosed in the "progress of modern biomedical science"; and Zhu Jianguo et al, in World Latest Medicine Information (Electronic Version) materials and methods disclosed in the "study of bacterial growth and capsular polysaccharide in Streptococcus pneumoniae fermentation process of 19A".
As an exemplary embodiment, resuscitating the strain in the solid medium may be performed according to the following method: inoculating the strain on solid culture medium with physiological saline, culturing in carbon dioxide incubator for 9-15 hr at carbon dioxide concentration of 10+ -0.5% and culture temperature of 36+ -0.1deg.C, and culturing as first generation.
The amplification culture in the liquid medium can be achieved by the means known to those skilled in the art. For example Chai Yanjing et al, in the "optimization of Streptococcus pneumoniae type 5 fermentation process" 1.4 culture section disclosed in the "progress of modern biomedical science"; and Zhu Jianguo et al in World Latest Medicine Information (Electronic Version) materials and methods disclosed in the "study of bacterial growth and capsular polysaccharide in Streptococcus pneumoniae fermentation process of 19A"; and Norma Sua' -rez in "Optimal Conditions for Streptococcus pneumoniae Culture: in Solid and Liquid Media".
As an exemplary embodiment, the amplification culture in a liquid medium may be performed according to the following method: transferring the strain into liquid culture medium after the first generation culture, placing into incubator for second generation culture, and collecting strain liquid OD 694 And (3) carrying out third-generation culture in a liquid culture medium at the time of 0.4-0.6, wherein the culture conditions of the second-generation culture and the third-generation culture are 36+/-0.1 ℃ and the carbon dioxide concentration is 10+/-0.5%.
The invention provides a feed medium for fermenting capsular polysaccharide from streptococcus pneumoniae 14, which comprises the following components: 180-220g/L glucose, 100-150g/L yeast extract, 100-150g/L soybean peptone, 1.1-1.5g/L amino acid, 0.3-0.8g/L magnesium salt, 0.05-0.2g/L ferrous salt, 0.01-0.02g/L zinc salt and 5-10g/L manganese salt.
The amino acids mentioned above include L-glutamine and asparagine.
Preferably, the feed medium further comprises choline chloride and a reducing agent.
Preferably, the feed medium comprises the following components: 180-220g/L glucose, 100-150g/L yeast extract, 100-150g/L soybean peptone, 0.46-0.52 g/L-glutamine, 0.7-0.9g/L asparagine and MgSO 4 ·7H 2 O 0.45-0.53g/L，FeSO 4 ·7H 2 O 0.09-0.11g/L，ZnSO 4 ·7H 2 O 0.015-0.017g/L，MnSO 4 ·H 2 O6.8-7.6 g/L, thioglycollic acid 0.18-0.22mL/L, choline chloride 0.01-0.03 g/L.
In some embodiments of the invention, the feed medium comprises the following components: 200+ -10g/L glucose, 100-150g/L yeast extract, 100-150g/L soybean peptone, 0.496+ -0.02 g/L-glutamine, 0.8+ -0.04 g/L asparagine, 0.02+ -0.001 g/L choline chloride, and MgSO 4 ·7H 2 O 0.5±0.025g/L，FeSO 4 ·7H 2 O 0.1±0.005g/L，ZnSO 4 ·7H 2 O 0.016±0.0008g/L，MnSO 4 ·H 2 O7.2+/-0.36 g/L, thioglycollic acid 0.2+/-0.01 ml/L.
The invention also provides application of the feed medium in fermenting capsular polysaccharide of streptococcus pneumoniae 14.
Based on the fermentation method, the invention provides an application of the fermentation method of the type 14 streptococcus pneumoniae in preparing streptococcus pneumoniae refined capsular polysaccharide.
The invention provides an application of the fermentation method of the 14 streptococcus pneumoniae in preparing streptococcus pneumoniae capsular polysaccharide degradation polysaccharide.
The invention provides an application of the fermentation method of the 14 streptococcus pneumoniae in preparing streptococcus pneumoniae capsular polysaccharide vaccines or streptococcus pneumoniae capsular polysaccharide conjugate vaccines.
In the invention, the streptococcus pneumoniae capsular polysaccharide vaccine or conjugate vaccine comprises a 23-valent streptococcus pneumoniae capsular polysaccharide vaccine, a 13-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine, a 20-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine and a 24-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine.
Wherein the 23-valent streptococcus pneumoniae capsular polysaccharide vaccine comprises capsular polysaccharides of 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F streptococcus pneumoniae serotypes.
The 13-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine comprises capsular polysaccharides of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae serotypes.
The 20-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine comprises capsular polysaccharides of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F streptococcus pneumoniae serotypes.
The 24-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine comprises capsular polysaccharides of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F streptococcus pneumoniae serotypes.
Based on the fermentation method, the invention provides a preparation method of 14 streptococcus pneumoniae capsular polysaccharide, which comprises the following steps: the fermentation method of the 14 type streptococcus pneumoniae is adopted to ferment the 14 type streptococcus pneumoniae, the fermentation liquor is sterilized after the fermentation is finished, the capsular polysaccharide in the fermentation liquor is collected, and the capsular polysaccharide is purified to obtain refined capsular polysaccharide, or the capsular polysaccharide degradation polysaccharide is obtained through purification and degradation.
As an exemplary embodiment, sodium Deoxycholate (DOC) is added in the later period of fermentation to sterilize and release capsular polysaccharide on the surface of streptococcus pneumoniae, the separated supernatant containing polysaccharide is subjected to ultrafiltration to remove small molecule residual substances, acid precipitation and alcohol precipitation to remove impurities such as protein nucleic acid, and finally ultrafiltration and freeze-drying are carried out to obtain refined polysaccharide.
The yield of the refined polysaccharide prepared by fermenting the streptococcus pneumoniae 14 by adopting the fermentation method is obviously higher than that of the conventional fermentation process, and the quality of the refined polysaccharide (including impurities such as protein and nucleic acid, quality control indexes such as specific groups and the like) can meet the national formulary requirements.
The beneficial effects of the invention at least comprise: the invention remarkably improves the capsular polysaccharide yield of the type 14 streptococcus pneumoniae by optimizing the feed supplement culture medium used in the fermentation process of the type 14 streptococcus pneumoniae. The feed medium used in the present invention is free of animal-derived materials, avoiding the risk that animal pathogens or other harmful materials may be contained therein. The fermentation method of the type 14 streptococcus pneumoniae provided by the invention can be used for preparing streptococcus pneumoniae capsular polysaccharide vaccines and polysaccharide conjugate vaccines.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The streptococcus pneumoniae 14 used in the following examples is obtained from the China medical Collection of bacteria, accession number CMCC 31608, and is approved by the national food and drug verification institute and is commercially available.
Example 1 optimization of feed Medium for Streptococcus pneumoniae capsular polysaccharide fermentation Process
The invention optimizes the feed medium of the fermentation method of the streptococcus pneumoniae capsular polysaccharide 14, and the following lists partial experimental results of the optimization of the feed medium.
The fermentation method of the 14-type streptococcus pneumoniae capsular polysaccharide adopted in the embodiment comprises the following steps:
1. resuscitating the strain onto solid culture medium with physiological saline, and culturing in carbon dioxide incubator for 9-15 hr (carbon dioxide concentration is 10+ -0.5% and culture temperature is 36+ -0.1deg.C) to obtain first generation culture;
2. transferring the strain into 120 mL liquid culture medium after the first generation culture, and placing into an incubator for second generation culture under the conditions of 36+ -0.1deg.C and 10+ -0.5% carbon dioxide concentration when the strain liquid OD 694 Performing third-generation culture at the time of 0.4-0.6, wherein the culture condition is that the temperature is 36+/-0.1 ℃ and the carbon dioxide concentration is 10+/-0.5%;
3. bacterial liquid OD of third generation culture 694 When the ratio is 0.4-0.6, transferring the mixture into a fermentation tank filled with a fermentation medium according to the inoculum size of 4% -7% to ferment, and in the fermentation process, when glucose is in a fermentation systemWhen the concentration of the glucose in the fermentation system is lower than 15g/L, continuously feeding by adopting a feeding culture medium, so that the glucose concentration in the fermentation system is maintained to be not lower than 5g/L; the fermentation culture gas environment is 0.1+/-0.005 VVM nitrogen; the fermentation temperature is 37+/-0.1 ℃, the pH is 7.2+/-0.1, and the fermentation fixed rotating speed is 90+/-5 rpm; adding DOC in the later fermentation period for sterilization, taking the supernatant of the lysate, centrifuging, and measuring the fermentation yield of capsular polysaccharide; purifying the sterilized fermentation liquor to obtain refined polysaccharide.
In the above method, the solid medium in step 1 may be a solid medium commonly used in the prior art for activating and recovering streptococcus pneumoniae, and the components include soybean peptone, tryptophan, tyrosine, cysteine hydrochloride, glucose, growth factors, agar powder, etc., and reference may be made to the solid medium of streptococcus pneumoniae disclosed in CN107760633 a.
The liquid medium in step 2 and the fermentation medium in step 3 are formulated as follows: glucose 20g/L, yeast extract 20g/L, soybean peptone 20g/L, dipotassium hydrogen phosphate 5g/L, sodium bicarbonate 1g/L, L-glutamine 0.062g/L, asparagine 0.1g/L, choline chloride 0.01g/L, mgSO 4 ·7H 2 O 0.05g/L，FeSO 4 ·7H 2 O 0.05g/L，ZnSO 4 ·7H 2 O 0.008g/L，MnSO 4 ·H 2 O0.004 g/L, thioglycollic acid 0.1ml/L.
Three test groups (001, 002, 003) were set, and the three groups were different in the feed medium used, and continuous feed was performed using 20% glucose solution (001), feed medium 1 (002), and feed medium 2 (003), respectively, and the composition of each feed medium was shown in table 1. The fermentation densities and capsular polysaccharide yields of the groups of Streptococcus pneumoniae type 14 are shown in Table 2.
Table 1 feed medium composition for three test groups
TABLE 2 Streptococcus pneumoniae fermentation Density and capsular polysaccharide production from example 1
Conclusion: the invention determines a fermentation mode of fed batch through a large number of evaluations, and continuously feeds in the fermentation process so as to maintain the full supply of carbon sources and nitrogen sources in a fermentation system, and examines the proportion of different nutritional ingredients, and determines the optimal formula of the fed culture medium, wherein the formula comprises the optimization of the nutritional ingredients in the fed culture medium, the optimization of the concentration and proportion of the carbon sources and nitrogen sources and amino acid, the optimization of the content of metal ions and other various ingredients. The optimization result of the feed medium shows that the feed medium 1 and the feed medium 2 can both remarkably improve the growth density of streptococcus pneumoniae and greatly improve the yield of capsular polysaccharide, wherein the lifting amplitude of the feed medium 2 is larger, and the yield of capsular polysaccharide using the feed medium can be improved by more than 1 time on the basis of the original process (the feed medium is 20% glucose).
Based on the formula of the feed medium 2, the formula design space is further determined through experimental design, and the result shows that when MnSO exists in the feed medium 4 ·H 2 When the O content is regulated to 5g/L, the capsular polysaccharide yield (the capsular polysaccharide content in the supernatant of the fermentation broth) is 1.55g/L; when the yeast powder content in the feed medium is up-regulated to 200g/L, the capsular polysaccharide yield is 1.25g/L; when the soybean peptone content in the feed medium was up-regulated to 200g/L, the capsular polysaccharide yield was 1.38g/L.
The experimental verification proves that the formula of the feed medium is finally determined as follows: 180-220g/L glucose, 100-150g/L yeast extract, 100-150g/L soybean peptone, 0.46-0.52 g/L-glutamine, 0.7-0.9g/L asparagine and MgSO 4 ·7H 2 O 0.45-0.53g/L，FeSO 4 ·7H 2 O 0.09-0.11g/L，ZnSO 4 ·7H 2 O 0.015-0.017g/L，MnSO 4 ·H 2 O6.8-7.6 g/L, thioglycollic acid 0.18-0.22mL/L, choline chloride 0.01-0.03 g/L.
Example 2 optimization of culture gas for Streptococcus pneumoniae capsular polysaccharide fermentation Process
The fermentation gas environment of the streptococcus pneumoniae capsular polysaccharide fermentation process of type 14 was continued to be optimised based on the optimal feed medium determined in example 1.
The fermentation method of the 14-type streptococcus pneumoniae capsular polysaccharide adopted in the embodiment comprises the following steps:
1. resuscitating the strain into solid culture medium with physiological saline, and culturing in carbon dioxide incubator for 9-15 hr (carbon dioxide concentration is 10+ -0.5%, and culture temperature is 36+ -0.1deg.C) to obtain first generation culture;
2. transferring the strain into 120 mL liquid culture medium after the first generation culture, and placing into an incubator for second generation culture under the conditions of 36+ -0.1deg.C and 10+ -0.5% carbon dioxide concentration when the strain liquid OD 694 Performing third-generation culture at the time of 0.4-0.6, wherein the culture condition is that the temperature is 36+/-0.1 ℃ and the carbon dioxide concentration is 10+/-0.5%;
3. bacterial liquid OD of third generation culture 694 When the ratio is 0.4-0.6, transferring the mixture into a fermentation tank filled with a fermentation medium according to the inoculum size of 4-7% for fermentation, and adopting a feed medium 2 for continuous feed supplement in the fermentation process to maintain the glucose concentration in a fermentation system to be more than 5g/L; the fermentation conditions are that the temperature is 37+/-0.1 ℃, the pH is 7.2+/-0.1, the fermentation fixed rotating speed is 90+/-5 rpm, and ventilation is carried out in the fermentation process; adding DOC in the later fermentation period for sterilization, taking the supernatant of the lysate, centrifuging, and measuring the yield of capsular polysaccharide; purifying the sterilized fermentation liquor to obtain refined polysaccharide.
The solid medium, liquid medium and fermentation medium used in the above method were the same as in example 1.
Three test groups (004, 005, 006) were provided, which were different only in the use of different culture gas environments, 0.1 VVM.+ -. 0.005 nitrogen, 0.1 VVM.+ -. 0.005 100% carbon dioxide, and 0.1 VVM 10.+ -. 0.5% carbon dioxide, respectively. The fermentation densities and capsular polysaccharide yields of the groups of Streptococcus pneumoniae type 14 are shown in Table 3.
TABLE 3 Streptococcus pneumoniae fermentation Density and capsular polysaccharide production from example 2
Conclusion: the different culture gas environments were studied using a 15L bioreactor, including the use of 10.+ -. 0.5% CO 2 ，100% CO 2 N 2 Fed-batch fermentations were performed separately. The results show that although streptococcus pneumoniae is a facultative anaerobe, different fermentation gas environments can have obvious influence on fermentation growth of streptococcus pneumoniae and biosynthesis of capsular polysaccharide, and the growth density of streptococcus pneumoniae can be effectively improved and the yield of capsular polysaccharide can be improved by providing nitrogen coverage for fermentation of streptococcus pneumoniae.
Example 3 optimization of fermentation pH of Streptococcus pneumoniae capsular polysaccharide fermentation Process
The fermentation method of the 14-type streptococcus pneumoniae capsular polysaccharide adopted in the embodiment comprises the following steps:
1. resuscitating the strain into solid culture medium with physiological saline, and culturing in carbon dioxide incubator for 9-15 hr (carbon dioxide concentration is 10+ -0.5%, and culture temperature is 36+ -0.1deg.C) to obtain first generation culture;
2. transferring the strain into 120 mL liquid culture medium after the first generation culture, and placing into an incubator for second generation culture under the conditions of 36+ -0.1deg.C and 10+ -0.5% carbon dioxide concentration when the strain liquid OD 694 Performing third-generation culture at the time of 0.4-0.6, wherein the culture condition is that the temperature is 36+/-0.1 ℃ and the carbon dioxide concentration is 10+/-0.5%;
3. bacterial liquid OD of third generation culture 694 When the ratio is 0.4-0.6, transferring the mixture into a fermentation tank filled with a fermentation medium according to the inoculum size of 4-7% for fermentation, and adopting a feed medium 2 for continuous feed supplement in the fermentation process to maintain the glucose concentration in a fermentation system to be more than 5g/L; the fermentation conditions are that the temperature is 37+/-0.1 ℃, the pH is 7.2+/-0.1, the fermentation fixed rotating speed is 90+/-5 rpm, and the culture gas is 0.1+/-0.005 VVM nitrogen; adding DOC in the later fermentation period for sterilization, taking the supernatant of the lysate, centrifuging, and measuring the yield of capsular polysaccharide; purifying the sterilized fermentation liquor to obtain refined polysaccharide.
The solid medium, liquid medium and fermentation medium used in the above method were the same as in example 1.
Four test groups (007, 008, 009, 010) were provided, which were different only in the pH conditions during fermentation, and pH 6.0.+ -. 0.1,6.4.+ -. 0.1,6.8.+ -. 0.1,7.2.+ -. 0.1 was used. The fermentation densities and capsular polysaccharide yields of the groups of Streptococcus pneumoniae type 14 are shown in Table 4.
TABLE 4 Streptococcus pneumoniae fermentation Density and capsular polysaccharide production from example 3
Conclusion: the fermentation adopts different pH values to influence the growth of streptococcus pneumoniae and the biosynthesis of polysaccharide, and the growth density of streptococcus pneumoniae and the yield of capsular polysaccharide can be effectively improved by providing the fermentation of streptococcus pneumoniae with the pH environment which is most suitable for bacterial growth and capsular polysaccharide synthesis. The results show that although pH 6.0-6.8 is effective in supporting high density growth of Streptococcus pneumoniae, the synthesis of capsular polysaccharides shows a large difference, ultimately selecting pH 6.8.+ -. 0.1 as the optimal fermentation pH.
Example 4 optimization of fermentation temperature of Streptococcus pneumoniae capsular polysaccharide fermentation Process
The fermentation method of the 14-type streptococcus pneumoniae capsular polysaccharide adopted in the embodiment comprises the following steps:
1. resuscitating the strain into solid culture medium with physiological saline, and culturing in carbon dioxide incubator for 9-15 hr (carbon dioxide concentration is 10+ -0.5%, and culture temperature is 36+ -0.1deg.C) to obtain first generation culture;
2. transferring the strain into 120 mL liquid culture medium after the first generation culture, and placing into an incubator for second generation culture under the conditions of 36+ -0.1deg.C and 10+ -0.5% carbon dioxide concentration when the strain liquid OD 694 Performing third-generation culture at the time of 0.4-0.6, wherein the culture condition is that the temperature is 36+/-0.1 ℃ and the carbon dioxide concentration is 10+/-0.5%;
3. bacterial liquid OD of third generation culture 694 When the inoculation amount is 0.4-0.6, the inoculation amount is 4-7% and the inoculation amount is transferred to the containerFermenting in a fermentation tank of a fermentation medium, and continuously feeding by adopting a feeding medium 2 in the fermentation process to maintain the glucose concentration in a fermentation system to be more than 5g/L; the fermentation conditions are that the temperature is 37+/-0.1 ℃ (different test groups are set according to the following temperatures), the pH is 6.8+/-0.1, the fermentation fixed rotating speed is 90+/-5 rpm, and the culture gas is 0.1+/-0.005 VVM nitrogen; adding DOC in the later fermentation period for sterilization, taking the supernatant of the lysate, centrifuging, and measuring the yield of capsular polysaccharide; purifying the sterilized fermentation liquor to obtain refined polysaccharide.
The solid medium, liquid medium and fermentation medium used in the above method were the same as in example 1.
Five test groups (011, 012, 013, 014, 015) were provided, differing only in the fermentation temperatures used, 35.+ -. 0.1 ℃, 36.+ -. 0.1 ℃, 37.+ -. 0.1 ℃, 38.+ -. 0.1 ℃ and 39.+ -. 0.1 ℃ respectively. The fermentation densities and capsular polysaccharide yields of the groups of Streptococcus pneumoniae type 14 are shown in Table 5.
TABLE 5 Streptococcus pneumoniae fermentation Density and capsular polysaccharide production from example 4
Conclusion: different fermentation temperatures can influence fermentation growth of streptococcus pneumoniae and polysaccharide biosynthesis, and the growth density of the streptococcus pneumoniae can be effectively improved and the yield of the capsular polysaccharide can be improved by providing a temperature environment which is most suitable for bacterial growth and capsular synthesis for fermentation of the streptococcus pneumoniae. The results show that when the fermentation temperature is between 36 ℃ and 39 ℃, although the growth curves of streptococcus pneumoniae are similar, the growth density can be higher, the synthesis of capsular polysaccharide is different, and finally the highest capsular polysaccharide yield can be obtained by selecting 37+/-0.1 ℃ as the optimal fermentation temperature.
Example 5 fermentation method and purification Process of Streptococcus pneumoniae capsular polysaccharide 14
Based on the optimized and determined optimal feed medium and optimal fermentation conditions, the fermentation production of the 14 Streptococcus pneumoniae capsular polysaccharide is carried out, and the fermentation method of the 14 Streptococcus pneumoniae capsular polysaccharide adopted in the embodiment comprises the following steps:
1. resuscitating the strain into solid culture medium with physiological saline, and culturing in carbon dioxide incubator for 9-15 hr (carbon dioxide concentration is 10+ -0.5%, and culture temperature is 36+ -0.1deg.C) to obtain first generation culture;
2. transferring the strain into 120 mL liquid culture medium after the first generation culture, and placing into an incubator for second generation culture under the conditions of 36+ -0.1deg.C and 10+ -0.5% carbon dioxide concentration when the strain liquid OD 694 Performing third-generation culture at the time of 0.4-0.6, wherein the culture condition is that the temperature is 36+/-0.1 ℃ and the carbon dioxide concentration is 10+/-0.5%;
3. bacterial liquid OD of third generation culture 694 When the ratio is 0.4-0.6, transferring the mixture into a fermentation tank filled with a fermentation medium according to the inoculum size of 4-7% for fermentation, and adopting a feed medium 2 for continuous feed supplement in the fermentation process to maintain the glucose concentration in a fermentation system to be more than 5g/L; the fermentation conditions are that the temperature is 37+/-0.1 ℃ (different test groups are set according to the following temperatures), the pH is 6.8+/-0.1, the fermentation fixed rotating speed is 90+/-5 rpm, and the culture gas is 0.1+/-0.005 VVM nitrogen; dividing the fermentation broth fermented to the late logarithmic growth stage into two parts, adding DOC into one part for sterilization, taking the supernatant of the lysate, centrifuging, measuring the yield of capsular polysaccharide, adding DOC into the other part of fermentation broth to a final concentration of 0.1%, standing at 2-8 ℃ for overnight, centrifuging the fermentation broth at 8000rpm for 30 min, and collecting the supernatant for purifying and preparing refined capsular polysaccharide.
The solid medium, liquid medium and fermentation medium used in the above method were the same as in example 1. The above-mentioned fermentations were carried out in parallel in three batches (016, 017, 018).
The purification method of the capsular polysaccharide comprises the following steps:
1. ultrafiltering and concentrating the collected supernatant by using a membrane bag with the aperture of 100KD to obtain concentrated solution, performing constant volume displacement on the concentrated solution, wherein the displacement solution is water for injection, the volume of the water for injection is 4-6 times of that of the concentrated solution, and the ultrafiltration concentrated solution is obtained;
2. adding glacial acetic acid into the ultrafiltration concentrated solution obtained in the previous step to adjust the pH to 3.8-4.2, fully stirring, standing at 2-8deg.C for more than 1 hr, and centrifuging to collect supernatant;
3. adjusting pH of the acid precipitation supernatant obtained in the previous step to 6.0-7.0 by using a 5M NaOH solution, adding disodium hydrogen phosphate to 10mmol/L of concentration, sodium dihydrogen phosphate (monohydrate) to 10mmol/L of concentration, sodium chloride to 0.15mol/L of concentration, anhydrous sodium acetate to 0.9mol/L of concentration and calcium chloride (dihydrate) to 0.15mol/L of concentration according to the volume of the feed liquid, adding glacial acetic acid to adjust pH to 5.3-5.5 after fully stirring uniformly, adding 30% (v/v) of absolute ethyl alcohol according to the volume of the feed liquid, standing for more than 3 hours at 2-8 ℃ after fully stirring uniformly, and centrifuging to collect the supernatant;
4. performing constant volume displacement on the supernatant obtained in the previous step by using a membrane bag with the aperture of 100KD, wherein the displacement solution is water for injection, the volume of the water for injection is more than 8 times of that of the supernatant, and concentrating the feed liquid to obtain capsular polysaccharide stock solution;
5. and freeze-drying the capsular polysaccharide solution, and collecting refined polysaccharide after freeze-drying.
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in tables 6 and 7.
TABLE 6 quality control indicators for Streptococcus pneumoniae refined capsular polysaccharide of example 5
TABLE 7 fermentation yield and recovery of Streptococcus pneumoniae refined polysaccharide of example 5
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Conclusion: in the embodiment, 3 batches of capsular polysaccharide of 14 streptococcus pneumoniae are fermented and purified continuously, and the quality of the prepared 14 streptococcus pneumoniae refined capsular polysaccharide meets the pharmacopoeia requirements. The result shows that after the feed culture medium and the fermentation condition are optimized, the fermentation yield of the capsular polysaccharide of the streptococcus pneumoniae 14 can reach the level of 2.4-2.6 g/L, and is improved by about 4 times compared with the fermentation method before optimization.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method of fermenting streptococcus pneumoniae type 14, the method comprising: in the fermentation process of the streptococcus pneumoniae type 14, feeding is carried out by a feeding culture medium;
the feed medium comprises the following components: 180-220g/L glucose, 100-150g/L yeast extract, 100-150g/L soybean peptone, 1.1-1.5g/L amino acid, 0.3-0.8g/L magnesium salt, 0.05-0.2g/L ferrous salt, 0.01-0.02g/L zinc salt and 5-10g/L manganese salt.
2. The method of fermentation of streptococcus pneumoniae of claim 1 wherein the amino acids comprise L-glutamine and asparagine;
and/or, the feed medium further comprises choline chloride and a reducing agent.
3. The fermentation process of streptococcus pneumoniae of claim 2 wherein the feed medium comprises the following components: 180-220g/L glucose, 100-150g/L yeast extract, 100-150g/L soybean peptone, 0.46-0.52 g/L-glutamine, 0.7-0.9g/L asparagine and MgSO 4 ·7H 2 O 0.45-0.53g/L，FeSO 4 ·7H 2 O 0.09-0.11g/L，ZnSO 4 ·7H 2 O 0.015-0.017g/L，MnSO 4 ·H 2 O6.8-7.6 g/L, thioglycollic acid 0.18-0.22mL/L, choline chloride 0.01-0.03 g/L.
4. A fermentation process for streptococcus pneumoniae type 14 according to any one of claims 1-3 wherein the feed is a continuous feed at a rate to maintain the concentration of glucose in the fermentation system at no less than 5 g/L.
5. A fermentation method of streptococcus pneumoniae type 14 according to any one of claims 1-3, wherein the gaseous environment of the fermentation is nitrogen.
6. The method according to claim 5, wherein nitrogen is introduced during the fermentation at an aeration rate of 0.08 to 0.12 VVM.
7. The method for fermenting streptococcus pneumoniae type 14 according to any one of claims 1-3 and 6, wherein the temperature of the fermentation is 36.5-37.5 ℃.
8. The fermentation method of streptococcus pneumoniae type 14 according to any one of claims 1-3 and 6, wherein the pH of the fermentation is 6.6-7.3;
and/or the rotational speed of the fermentation is 80-100rpm.
9. Use of the fermentation process of streptococcus pneumoniae type 14 according to any of claims 1-8 for any of the following:
(1) The application in preparing streptococcus pneumoniae refined capsular polysaccharide;
(2) The application of the streptococcus pneumoniae capsular polysaccharide degradation polysaccharide in preparation of the streptococcus pneumoniae capsular polysaccharide;
(3) The application of the streptococcus pneumoniae capsular polysaccharide vaccine or the streptococcus pneumoniae capsular polysaccharide conjugate vaccine in preparation.
10. A method of preparing a streptococcus pneumoniae capsular polysaccharide of type 14, the method comprising: the method for fermenting streptococcus pneumoniae type 14 according to any one of claims 1-8, wherein the streptococcus pneumoniae type 14 is fermented, the fermentation broth is sterilized after the fermentation is completed, capsular polysaccharide is collected and purified to obtain refined capsular polysaccharide, or capsular polysaccharide degradation polysaccharide is obtained through purification and degradation.
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