Chemically defined culture medium for producing outer membrane vesicles and use thereof

Abstract

The present invention discloses a chemically defined culture medium for producing outer membrane vesicles and its use. Specifically, the chemically defined culture medium of the present invention comprises: 0.75-12 g / L ammonium sulfate, 2.5-20 g / L sodium chloride, 0.75-12 g / L sodium hydrogen phosphate, 0.75-12 g / L sodium thiosulfate pentahydrate, 2.5-40 g / L glucose, 0.075-1.2 g / L glutamic acid, 0.075-1.2 g / L arginine, 0.075-1.2 g / L serine, 0.001-0.02 g / L vitamin B5, 0.001-0.02 g / L vitamin B6, 0.01-0.2 g / L calcium chloride dihydrate, 0.03-0.4 g / L magnesium sulfate, and 0.003-0.04 g / L ferrous sulfate heptahydrate. The chemically defined culture medium of the present invention is safe, environmentally friendly, and can produce a high yield of outer membrane vesicles.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial culture, in particular to a chemically defined culture medium for producing outer membrane vesicles and application thereof. Background Art

[0002] Outer membrane vesicles (OMVs) are membranous nanostructures produced by Gram-negative bacteria. They are highly immunogenic, non-infectious, and non-replicative, making them suitable for vaccine production. By adding heterologous antigens to OMVs, antigen expression can be maintained without altering their properties, expanding their application as vaccine components and establishing an OMV-based vaccine platform. However, not all Gram-negative bacteria are suitable for OMV vaccine production due to their high pathogenicity, slow growth rate, complex culture requirements, and low OMV yield.

[0003] Neisseria meningitidis (N. meningitidis), also known as meningococcus, is the causative agent of epidemic cerebrospinal meningitis (EMM). Meningococci are classified into five distinct serotypes: A, B, C, W, and Y, based on the structure of their capsular polysaccharides. Clinical studies have shown that capsular polysaccharides from serotypes A, C, W, and Y can produce a strong immune response, leading to the introduction of polysaccharide vaccines for serotypes AC, W, and Y in the 1980s. However, due to the low immunogenicity of serotype B capsular polysaccharides, vaccine development for serotype B meningitis has been slow. Against this backdrop, global outbreaks of serotype B meningitis have led to the development of several OMV vaccines. For example, the vaccines VA-MENGOCOC-BC® launched in Cuba, MenBVac® launched in Norway, and MeNZB® launched in New Zealand have all demonstrated their effectiveness against serotype B meningitis.

[0004] However, in the process of producing safer, more effective, and cost-effective OMV vaccines, researchers have discovered that many process challenges remain to be overcome. Meningococci are fastidious bacteria with very high requirements for culture media. Complex media (such as trypticase soy peptone) are typically used, but these media have numerous drawbacks. First, they may contain animal-derived or allergens, posing safety risks and should be avoided in the production of human pharmaceuticals. Second, the complex composition of the complexes makes it difficult to maintain batch-to-batch consistency. Finally, the complexes are typically hygroscopic powders, which are prone to adsorption during weighing, resulting in uneven weighing. The dissolution process can also generate dust, causing uneven weight and polluting the production environment.

[0005] Therefore, there is an urgent need to develop a chemically defined medium (CDM) with clearly controlled components and no animal origin. Summary of the Invention

[0006] The purpose of the present invention is to provide a chemically defined culture medium for producing outer membrane vesicles and use thereof.

[0007] In one aspect, the present invention provides a chemically defined medium comprising: 0.75-12 g / L ammonium sulfate, 2.5-20 g / L sodium chloride, 0.75-12 g / L disodium hydrogen phosphate, 0.75-12 g / L sodium thiosulfate pentahydrate, 2.5-40 g / L glucose, 0.075-1.2 g / L glutamic acid, 0.075-1.2 g / L arginine, 0.075-1.2 g / L serine, 0.001-0.02 g / L vitamin B5, 0.001-0.02 g / L vitamin B6, 0.01-0.2 g / L calcium chloride dihydrate, 0.03-0.4 g / L magnesium sulfate, and 0.003-0.04 g / L ferrous sulfate heptahydrate.

[0008] In one embodiment, the culture medium comprises: 1.5 g / L ammonium sulfate, 5 g / L sodium chloride, 3 g / L sodium phosphate dibasic, 6 g / L sodium thiosulfate pentahydrate, 20 g / L glucose, 0.6 g / L glutamic acid, 1.2 g / L arginine, 0.3 g / L serine, 0.005 g / L vitamin B5, 0.001 g / L vitamin B6, 0.1 g / L calcium chloride dihydrate, 0.4 g / L magnesium sulfate, and 0.04 g / L ferrous sulfate heptahydrate.

[0009] In one embodiment, the culture medium comprises: 3 g / L ammonium sulfate, 10 g / L sodium chloride, 3 g / L sodium phosphate dibasic, 3 g / L sodium thiosulfate pentahydrate, 10 g / L glucose, 0.3 g / L glutamic acid, 0.3 g / L arginine, 0.3 g / L serine, 0.005 g / L vitamin B5, 0.005 g / L vitamin B6, 0.05 g / L calcium chloride dihydrate, 0.1 g / L magnesium sulfate, and 0.01 g / L ferrous sulfate heptahydrate.

[0010] In one embodiment, the culture medium comprises: 0.75 g / L ammonium sulfate, 2.5 g / L sodium chloride, 0.75 g / L sodium phosphate dibasic, 0.75 g / L sodium thiosulfate pentahydrate, 2.5 g / L glucose, 0.075 g / L glutamic acid, 0.075 g / L arginine, 0.075 g / L serine, 0.001 g / L vitamin B5, 0.001 g / L vitamin B6, 0.01 g / L calcium chloride dihydrate, 0.03 g / L magnesium sulfate, and 0.003 g / L ferrous sulfate heptahydrate.

[0011] In one embodiment, the culture medium comprises: 12 g / L ammonium sulfate, 20 g / L sodium chloride, 12 g / L sodium phosphate dibasic, 12 g / L sodium thiosulfate pentahydrate, 40 g / L glucose, 1.2 g / L glutamic acid, 1.2 g / L arginine, 1.2 g / L serine, 0.02 g / L vitamin B5, 0.02 g / L vitamin B6, 0.2 g / L calcium chloride dihydrate, 0.4 g / L magnesium sulfate, and 0.04 g / L ferrous sulfate heptahydrate.

[0012] In one embodiment, the culture medium comprises: 1 g / L ammonium sulfate, 6 g / L sodium chloride, 5 g / L sodium hydrogen phosphate, 3 g / L sodium thiosulfate pentahydrate, 15 g / L glucose, 1 g / L glutamic acid, 0.5 g / L arginine, 1 g / L serine, 0.01 g / L vitamin B5, 0.005 g / L vitamin B6, 0.18 g / L calcium chloride dihydrate, 0.2 g / L magnesium sulfate, 0.02 g / L ferrous sulfate heptahydrate,

[0013] In one embodiment, the culture medium comprises: 3 g / L ammonium sulfate, 10 g / L sodium chloride, 10 g / L sodium phosphate dibasic, 5 g / L sodium thiosulfate pentahydrate, 10 g / L glucose, 0.5 g / L glutamic acid, 1 g / L arginine, 1 g / L serine, 0.01 g / L vitamin B5, 0.01 g / L vitamin B6, 0.1 g / L calcium chloride dihydrate, 0.1 g / L magnesium sulfate, and 0.01 g / L ferrous sulfate heptahydrate.

[0014] In one embodiment, the culture medium comprises: 2 g / L ammonium sulfate, 8 g / L sodium chloride, 5 g / L sodium phosphate dibasic, 6 g / L sodium thiosulfate pentahydrate, 20 g / L glucose, 1 g / L glutamic acid, 0.8 g / L arginine, 0.8 g / L serine, 0.03 g / L vitamin B5, 0.01 g / L vitamin B6, 0.1 g / L calcium chloride dihydrate, 0.1 g / L magnesium sulfate, and 0.01 g / L ferrous sulfate heptahydrate.

[0015] In one aspect, the present invention provides a method for producing bacterial outer membrane vesicles, comprising the steps of: (i) culturing bacteria using a culture medium as described in the present invention under conditions suitable for producing outer membrane vesicles; (ii) harvesting the produced outer membrane vesicles; and optionally (iii) purifying the outer membrane vesicles.

[0016] In one embodiment, the bacterium is N. meningitidis.

[0017] In one embodiment, the meningococcus is a natural and / or genetically modified serogroup B meningococcus. In one embodiment, the meningococcus is a natural MC58 and / or a genetically modified MC58 strain. In one embodiment, the meningococcus is a natural MC58 and / or an MC58 strain that does not express the rmpM gene. In one embodiment, the meningococcus is a natural MC58 strain and / or an MC58 strain in which the rmpM gene is knocked out.

[0018] In one aspect, the present invention provides a method for preparing a vaccine, comprising the steps of: (i) culturing meningococci using a culture medium as described in the present invention under culture conditions suitable for producing outer membrane vesicles; (ii) harvesting the produced outer membrane vesicles; and (iii) adding a pharmaceutically acceptable carrier and / or adjuvant to prepare a vaccine.

[0019] In one embodiment, after step (ii), the method further comprises the step of purifying the outer membrane vesicles.

[0020] In one embodiment, the meningococcus is serogroup B meningococcus. In one embodiment, the meningococcus is MC58. In one embodiment, the meningococcus is MC58 that does not express rmpM.

[0021] In one aspect, the present invention provides use of the culture medium of the present invention in producing outer membrane vesicles or preparing vaccines.

[0022] In one specific embodiment, the present invention provides a high-yield OMV CDM medium (chemically defined medium). Through a rigorous formulation, this medium rapidly grows meningococci to high OD concentrations, slows aging, prolongs the stable period, significantly increases OMV yields, and reduces residual nucleic acids compared to other known media for culturing meningococci. The inventors developed this CDM medium for meningococci through in-depth research on meningococcal metabolic pathways, rationally selected appropriate compound components, and extensive formulation optimization experiments. The formulation of this medium is free of complexes such as tryptone or soy peptone, has a well-defined composition, and eliminates animal-derived or allergenic risks. It utilizes pharmaceutical-grade excipients with excellent batch-to-batch consistency. It contains no powdered ingredients, eliminating the risk of moisture absorption and dust generation. The formulation is easily soluble, eliminating the need for defoaming agents. It is colorless, transparent, and pigment-free, facilitating downstream purification. It can increase fermentation OD, slow bacterial aging, increase yield, and reduce residual nucleic acids.

[0023] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0024] The reagents and raw materials used in the present invention are commercially available.

[0025] The positive progress effect of the present invention is:

[0026] (1) The CDM culture medium of the present invention does not contain any complexes, has clear ingredients, has no animal origin or allergenic risks, and has good safety.

[0027] (2) The CDM culture medium of the present invention is composed of pharmaceutical-grade compounds, and the excipients have good batch-to-batch consistency; it has no powder components, is non-hygroscopic, and does not generate dust.

[0028] (3) The CDM culture medium of the present invention is also characterized by rapid dissolution, does not require the addition of defoaming agents, is colorless and transparent, and does not contain pigments, which is more conducive to the growth of pathogens. Since no defoaming agent is added, there is no need to detect defoaming agent residues in downstream processing.

[0029] (4) The fermentation product has a higher OD (can reach 9.5 after culture), which is 1.3 times higher than TSB (trypticase soy broth) composite culture medium.

[0030] (5) The present invention can achieve a higher OMV yield, which is 1.49 times higher than that of TSB composite culture medium.

[0031] (6) Lower residual nucleic acids. Since the composite culture medium initially contains a certain amount of nucleic acids, while the CDM culture medium of the present invention has zero initial nucleic acid content, bacterial aging can be slowed down, further reducing the release of nucleic acids caused by bacterial rupture. The residual nucleic acids in the CDM culture medium of the present invention are reduced by 55% compared to the TSB composite culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The OD and nucleic acid residue results of the strains grown in fermenter TSB and CDM medium are shown.

[0033] Figure 2 The OD and nucleic acid residue results of the strains cultured in CDM1 and CDM2 medium are shown.

[0034] Figure 3 The OD and nucleic acid residue results of the strains cultured in CDM3 and CDM4 medium are shown.

[0035] Figure 4 The OD and nucleic acid residue results of MC58 and MC58ΔrmpM fermentations are shown. DETAILED DESCRIPTION

[0036] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0037] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0038] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0039] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, normal saline, lactated Ringer's solution, standard sucrose, standard dextrose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, saline solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose), amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and colorants.

[0040] Explanation of terms

[0041] Composite culture medium: A culture medium containing a complex, generally using animal protein hydrolysate or plant protein hydrolysate. The culture medium has complex ingredients and unclear composition, and is rich in vitamins, amino acids and other substances that are beneficial to fermentation.

[0042] CDM culture medium: also known as chemically defined culture medium, means that all ingredients in the culture medium are clear, and it does not contain animal protein or plant hydrolysates. Instead, it uses some small molecule compounds with known structures and functions, such as amino acids, short peptides, plant hormones, etc.

[0043] TSB medium: Trypticase soy broth, a universal nutrient medium used for the cultivation of various microorganisms, is a composite medium.

[0044] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0045] The examples of the present invention use natural meningococcal strain MC58 or meningococcal strain MC58ΔrmpM with the rmpM gene knocked out, which is only used to illustrate the invention and is not intended to limit the scope of the invention. Obviously, various modifications and variations can be made to the present invention without departing from the essence of the invention, and such modifications and variations are also within the scope of the invention.

[0046] Example 1: Steps for culturing meningococci in shake flasks

[0047] 1.1 Activation of glycerol tube-coated plates

[0048] Glycerol tubes of frozen meningococcal MC58 (purchased from ATCC) and meningococcal MC58ΔrmpM (see patent publication CN118240723B) that do not express the rmpM gene were taken from a -80°C freezer. In a biological safety cabinet (Thermo), the bacteria in the glycerol tubes were spread onto GC agar medium (OXOID) plates and incubated in a controlled carbon dioxide incubator (STIK) until an appropriate number of colonies grew on the plates.

[0049] 1.2 Shake flask seed culture

[0050] The colonies on the plate were inoculated into shake flasks. The shake flask culture medium used was TSB medium (BD), the pH was adjusted to 6.5-8, and after sterilization and cooling, glucose was added to a concentration of 3 g / L. The shake flasks were placed in a 37°C constant temperature shaking incubator (Minquan) for 5-6 hours.

[0051] 1.3 CDM shake flask culture

[0052] CDM culture medium was added to the shake flask, and the culture medium volume was controlled to 20% of the shake flask volume. The initial OD of the strain after inoculation was 0.05-0.3. The temperature was controlled at 33-39°C, the rotation speed was 150-250 rpm, and the culture was cultured in a constant temperature shaking incubator (Minquan) for 16 hours.

[0053] Example 2: CDM culture medium screening

[0054] 2.1 Determination of basal medium (BM) components

[0055] As is well known, CDM is a culture medium formed by adding appropriate amounts of amino acids, vitamins, and trace elements. Based on experience in microbial culture, a basal medium (BM) has been established, with the formula shown in Table 1.

[0056] The culture medium was prepared according to Table 1, the glycerol tube coating plate activation was completed according to 1.1 of Example 1, the shake flask seed culture was completed according to 1.2, and the CDM medium shake flask culture was carried out according to 1.3.

[0057] After culturing in a shaker for 16 h, the OD of the strain MC58ΔrmpM increased from the initial 0.1 to about 0.5, proving that the strain can grow in BM.

[0058] Table 1: BM culture medium

[0059]

[0060] 2.2 Determination of amino acids

[0061] Additional components of the culture medium were added according to Table 2, glycerol tube coating plate activation was completed according to 1.1 of Example 1, shake flask seed culture was completed according to 1.2, and CDM medium shake flask culture was performed according to 1.3.

[0062] Using BM as the control medium (OD 0.51 after 16 h of culture), a single-factor experiment with 16 amino acids (filter sterilized) was conducted. The results are shown in Table 2. It can be confirmed that the addition of glutamate, arginine, and serine is beneficial to the growth of the strain.

[0063] Table 2: Effects of different amino acids on strain growth

[0064]

[0065] 2.3 Determination of vitamins and trace elements

[0066] Additional components of the culture medium were added according to Table 3, glycerol tube coating plate activation was completed according to 1.1 of Example 1, shake flask seed culture was completed according to 1.2, and CDM medium shake flask culture was performed according to 1.3.

[0067] Using BM as the control medium (OD 0.51 after 16 h of culture), a single-factor experiment (filter sterilization) of 19 vitamins and trace elements was conducted. The results are shown in Table 3. It can be confirmed that the addition of vitamin B5, vitamin B6, calcium chloride dihydrate, magnesium sulfate, and ferrous sulfate heptahydrate is beneficial to the growth of the strain.

[0068] Table 3: Effects of different amino acids and trace elements on strain growth

[0069]

[0070] 2.4 Determination of BM+ culture medium

[0071] Additional components of the culture medium were added according to Table 4, glycerol tube coating plate activation was completed according to 1.1 of Example 1, shake flask seed culture was completed according to 1.2, and CDM medium shake flask culture was performed according to 1.3.

[0072] According to the results in Tables 2 and 3, amino acids, vitamins, and trace elements were added to BM to form a new medium BM+, the formula of which is shown in Table 4. After 16 h of culture in a shaker, the OD increased from the initial 0.1 to approximately 1.2.

[0073] Table 4: BM+ culture medium

[0074]

[0075] 2.5 Determination of the amount of each component added to BM+ medium

[0076] Add components of different concentrations to the culture medium according to Table 5, complete glycerol tube coating plate activation according to 1.1 of Example 1, complete shake flask seed culture according to 1.2, and perform CDM medium shake flask culture according to 1.3.

[0077] Using BM+ as the control medium (OD 1.23 after 16 hours of culture), the concentration of only one component in the culture medium was varied at a time. Using the concentration in BM+ as the central value, the concentrations were reduced by 1 / 2, 1 / 4, and increased by 2-fold and 4-fold to create five additive gradients. OD values were measured after 16 hours of culture. Experiments with the addition of 13 components were performed, and the results are shown in Table 5.

[0078] Table 5: OD values at different addition levels

[0079]

[0080] 2.6 Determination of CDM culture medium

[0081] The optimal addition concentration in Table 5 was selected to form CDM culture medium, and the formula was shown in Table 6.

[0082] Table 6: CDM medium

[0083]

[0084] Preparation method: Weigh according to the proportion, adjust the volume to the target volume, and filter and sterilize.

[0085] Example 3: Experimental steps for fermentation of meningococci

[0086] 3.1 Activation of glycerol tube coated plates.

[0087] Glycerol tubes of frozen meningococcal MC58 or MC58ΔrmpM were taken from a -80°C freezer. In a biological safety cabinet (Thermo), the bacteria in the glycerol tubes were spread onto GC agar medium (OXOID) plates and incubated in a controlled carbon dioxide incubator (STIK) until an appropriate number of colonies grew on the plates.

[0088] 3.2 Inoculation and expansion of shake flask culture

[0089] The colonies on the plate were inoculated into shake flasks. The shake flask culture medium used was TSB medium (BD), the pH was adjusted to 6.5-8, and after sterilization and cooling, glucose was added to a concentration of 3 g / L. The shake flasks were placed in a 37°C constant temperature shaking incubator (Minquan) for 5-6 hours.

[0090] 3.3 Fermentation tank culture

[0091] Sterilized tryptic soy casein broth (TSB) or CDM medium was added to a 5L dual fermenter (Baoxing-BIOTECH5JG). The medium volume was controlled at 40%-80% of the fermenter volume. TSB medium was added with a defoamer (Dow-DF104) at a volume ratio of 0.02%-0.05%, and CDM medium was not added with a defoamer. The initial OD of the strain after inoculation was 0.05-0.3. The temperature was controlled at 33-39°C, pH 6.5-8, air volume 0.4-2 vvm, dissolved oxygen 10%-60%, and the rotation speed was linked to the dissolved oxygen. Glucose was provided by the fermenter's feed peristaltic pump, and the culture was harvested after 18 hours of fermentation.

[0092] Example 4: Comparison of fermentation between CDM medium and TSB medium

[0093] The culture medium was prepared according to Table 6. The activation of the MC58ΔrmpM strain by glycerol tube coating was completed according to 3.1 of Example 3. The inoculation and expansion of the shake flask were completed according to 3.2. The fermenter culture was carried out according to 3.3.

[0094] The results are as follows Figure 1 As shown in Table 7. CDM medium grew slower than TSB medium in the first 0-4 hours, but after 4 hours, the growth rate quickly caught up with TSB medium and exceeded TSB after 10 hours. In particular, the OD value could be maintained at a high point for a longer time, avoiding bacterial cell breakage and producing less nucleic acid residue.

[0095] At harvest, the OD of CDM medium was 1.3 times higher than that of TSB medium, the residual nucleic acid was 55% lower, the average particle size of OMVs was similar, and the OMV particle concentration was 1.49 times higher.

[0096] Table 7: CDM and TSB harvest fluid testing

[0097]

[0098] Example 5: End value test of each component content in CDM culture medium

[0099] The culture medium was prepared according to Tables 8 and 9, respectively. The MC58ΔrmpM strain was activated by glycerol tube coating according to 3.1 of Example 3. The inoculation and shake flask expansion culture was completed according to 3.2. The fermentor culture was carried out according to 3.3.

[0100] Table 8: CDM1 medium

[0101]

[0102] Table 9: CDM2 medium

[0103]

[0104] The results are as follows Figure 2 As shown in Table 10, the growth rate of CDM1 medium was faster than that of CDM medium 2. The strain grew slowly at the two end values, with OD only reaching about 4, and less nucleic acid residue.

[0105] At harvest, the OD of CDM1 medium was slightly higher than that of CDM2 medium, the residual nucleic acid was 1.31 times higher, the average particle size of OMVs was similar, and the OMV particle concentration was 1.36 times higher.

[0106] Table 10: CDM1 and CDM2 harvest fluid assays

[0107]

[0108] Example 6: Random value test of the content of each component in CDM culture medium

[0109] The culture medium was prepared according to Table 11 and Table 12, respectively. The MC58ΔrmpM strain was activated by glycerol tube coating according to 3.1 of Example 3. The inoculation and shake flask expansion culture was completed according to 3.2. The fermentor culture was carried out according to 3.3.

[0110] Table 11: CDM3 medium

[0111]

[0112] Table 12: CDM4 medium

[0113]

[0114] The results are as follows Figure 3 As shown in Table 13, the growth rate of CDM3 medium was faster than that of CDM4 medium, the OD could reach above 6, and less nucleic acid residue was found.

[0115] At harvest, the OD of CDM3 medium was higher than that of CDM4 medium, the residual nucleic acid was 1.16 times higher, the average particle size of OMVs was similar, and the OMV particle concentration was 1.18 times higher.

[0116] Table 13: CDM3 and CDM4 harvest fluid assays

[0117]

[0118] Example 7: CDM medium for culture test of different strains

[0119] The culture medium was prepared according to Table 14. The activation of MC58 and MC58ΔrmpM strains by glycerol tube coating plates was completed according to 3.1 of Example 3. The inoculation and expansion of shake flasks were completed according to 3.2. The fermentor culture was carried out according to 3.3.

[0120] Table 14: CDM5 medium

[0121]

[0122] The fermentation results of the two strains are as follows Figure 4 As shown, strain MC58 grew faster than strain MC58ΔrmpM in CDM5 medium. At harvest, strain MC58 had a higher OD, but strain MC58ΔrmpM had 1.34-fold more residual nucleic acid, a 10 nm smaller average OMV size, and a 3.43-fold higher OMV concentration than strain MC58.

[0123] Table 15: Detection of strain MC58 and strain MC58ΔrmpM harvest fluid

[0124]

[0125] From the results in Table 15, it can be seen that CDM medium can achieve higher OD values for the cultivation of strains MC58 and MC58ΔrmpM, with low residual nucleic acid and high OMV yield.

Claims

1. A chemically defined medium, characterized in that The culture medium is composed of the following components: 0.75-12 g / L ammonium sulfate, 2.5-20 g / L sodium chloride, 0.75-12 g / L disodium hydrogen phosphate, 0.75-12 g / L sodium thiosulfate pentahydrate, 2.5-40 g / L glucose, 0.075-1.2 g / L glutamic acid, 0.075-1.2 g / L arginine, 0.075-1.2 g / L serine, 0.001-0.02 g / L vitamin B5, 0.001-0.02 g / L vitamin B6, 0.01-0.2 g / L calcium chloride dihydrate, 0.03-0.4 g / L magnesium sulfate, and 0.003-0.04 g / L ferrous sulfate heptahydrate.

2. The culture medium according to claim 1, wherein The culture medium consists of the following components: 1.5 g / L ammonium sulfate, 5 g / L sodium chloride, 3 g / L sodium hydrogen phosphate, 6 g / L sodium thiosulfate pentahydrate, 20 g / L glucose, 0.6 g / L glutamic acid, 1.2 g / L arginine, 0.3 g / L serine, 0.005 g / L vitamin B5, 0.001 g / L vitamin B6, 0.1 g / L calcium chloride dihydrate, 0.4 g / L magnesium sulfate, and 0.04 g / L ferrous sulfate heptahydrate.

3. The culture medium according to claim 1, wherein The culture medium consists of the following components: 3 g / L ammonium sulfate, 10 g / L sodium chloride, 3 g / L sodium hydrogen phosphate, 3 g / L sodium thiosulfate pentahydrate, 10 g / L glucose, 0.3 g / L glutamic acid, 0.3 g / L arginine, 0.3 g / L serine, 0.005 g / L vitamin B5, 0.005 g / L vitamin B6, 0.05 g / L calcium chloride dihydrate, 0.1 g / L magnesium sulfate, and 0.01 g / L ferrous sulfate heptahydrate.

4. The culture medium according to claim 1, wherein The culture medium consists of the following components: 0.75 g / L ammonium sulfate, 2.5 g / L sodium chloride, 0.75 g / L sodium hydrogen phosphate, 0.75 g / L sodium thiosulfate pentahydrate, 2.5 g / L glucose, 0.075 g / L glutamic acid, 0.075 g / L arginine, 0.075 g / L serine, 0.001 g / L vitamin B5, 0.001 g / L vitamin B6, 0.01 g / L calcium chloride dihydrate, 0.03 g / L magnesium sulfate, and 0.003 g / L ferrous sulfate heptahydrate.

5. The culture medium according to claim 1, wherein The culture medium consists of the following components: 12 g / L ammonium sulfate, 20 g / L sodium chloride, 12 g / L disodium hydrogen phosphate, 12 g / L sodium thiosulfate pentahydrate, 40 g / L glucose, 1.2 g / L glutamic acid, 1.2 g / L arginine, 1.2 g / L serine, 0.02 g / L vitamin B5, 0.02 g / L vitamin B6, 0.2 g / L calcium chloride dihydrate, 0.4 g / L magnesium sulfate, and 0.04 g / L ferrous sulfate heptahydrate.

6. The culture medium according to claim 1, wherein The culture medium consists of the following components: 1 g / L ammonium sulfate, 6 g / L sodium chloride, 5 g / L disodium hydrogen phosphate, 3 g / L sodium thiosulfate pentahydrate, 15 g / L glucose, 1 g / L glutamic acid, 0.5 g / L arginine, 1 g / L serine, 0.01 g / L vitamin B5, 0.005 g / L vitamin B6, 0.18 g / L calcium chloride dihydrate, 0.2 g / L magnesium sulfate, and 0.02 g / L ferrous sulfate heptahydrate.

7. The culture medium according to claim 1, wherein The culture medium consists of the following components: 3 g / L ammonium sulfate, 10 g / L sodium chloride, 10 g / L disodium hydrogen phosphate, 5 g / L sodium thiosulfate pentahydrate, 10 g / L glucose, 0.5 g / L glutamic acid, 1 g / L arginine, 1 g / L serine, 0.01 g / L vitamin B5, 0.01 g / L vitamin B6, 0.1 g / L calcium chloride dihydrate, 0.1 g / L magnesium sulfate, and 0.01 g / L ferrous sulfate heptahydrate.

8. The culture medium according to claim 1, wherein The culture medium consists of the following components: 2 g / L ammonium sulfate, 8 g / L sodium chloride, 5 g / L sodium hydrogen phosphate, 6 g / L sodium thiosulfate pentahydrate, 20 g / L glucose, 1 g / L glutamic acid, 0.8 g / L arginine, 0.8 g / L serine, 0.03 g / L vitamin B5, 0.01 g / L vitamin B6, 0.1 g / L calcium chloride dihydrate, 0.1 g / L magnesium sulfate, and 0.01 g / L ferrous sulfate heptahydrate.

9. A method for producing bacterial outer membrane vesicles, characterized in that: The method comprises the steps of: (i) culturing bacteria using the culture medium according to any one of claims 1 to 8 under conditions suitable for producing outer membrane vesicles; (ii) harvesting the produced outer membrane vesicles; and Optionally (iii) purifying the outer membrane vesicles; Wherein, the bacterium is meningococcus ( N.meningitidis )MC58.

10. A method for preparing a vaccine, characterized in that: The method comprises the steps of: (i) culturing meningococcal MC58 using the culture medium according to any one of claims 1 to 8 under culture conditions suitable for producing outer membrane vesicles; (ii) harvesting the produced outer membrane vesicles of meningococcal MC58; and (iii) adding a pharmaceutically acceptable carrier and / or adjuvant to prepare a vaccine.

11. Use of the culture medium according to any one of claims 1 to 8 in producing outer membrane vesicles or preparing a vaccine by producing outer membrane vesicles, wherein the outer membrane vesicles are outer membrane vesicles of meningococcus MC58.

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